Antiangiogenic peptides and methods for inhibiting angiogenesis

ABSTRACT

Mammalian kringle 5 peptide fragments are disclosed for treating angiogenic diseases Methods and compositions for inhibiting angiogenic diseases are also disclosed.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/643219 filed May 3, 1996, now U.S. Pat. No. 5,801,146.

TECHNICAL FIELD

The present invention relates to the field of peptide chemistry. Moreparticularly, the invention relates to the preparation and use ofpeptides containing amino acid sequences substantially similiar to thecorresponding sequences of the kringle 5 region of mammalianplasminogen, pharmaceutical compositions containing the peptides,antibodies specific for the angiostatin receptor, means for angiostatindetection and measurement, cytotoxic agents linked to angiostatinproteins and treatment of diseases which arise from or are exacerbatedby angiogenesis.

BACKGROUND OF THE INVENTION

Angiogenesis, the process by which new blood vessels are formed, isessential for normal body activities including reproduction, developmentand wound repair. Although the process is not completely understood, itis believed to involve a complex interplay of molecules which regulatethe growth of endothelial cells (the primary cells of capillary bloodvessels). Under normal conditions, these molecules appear to maintainthe microvasculature in a quiescent state (i.e. one of no capillarygrowth) for prolonged periods which may last for as long as weeks or, insome cases, decades. When necessary (such as during wound repair), thesesame cells can undergo rapid proliferation and turnover within a 5 dayperiod (Folkman, J. and Shing, Y., The Journal of Biological Chemistry,267 (16), 10931-10934, and Folkman, J. and Klagsbrun, M., Science, 235,442-447 (1987).

Although angiogenesis is a highly regulated process under normalconditions, many diseases (characterized as angiogenic diseases) aredriven by persistent unregulated angiogenesis. Otherwise stated,unregulated angiogenesis may either cause a particular disease directlyor exascerbate an existing pathological condition. For example, ocularneovacularization has been implicated as the most common cause ofblindness and dominates approximately 20 eye diseases. In certainexisting conditions, such as arthritis, newly formed capillary bloodvessels invade the joints and destroy cartilage. In diabetes, newcapillaries formed in the retina invade the vitreous, bleed, and causeblindness. Growth and metastasis of solid tumors are also dependent onangiogenesis (Folkman, J., Cancer Research, 46, 467-473 (1986), Folkman,J., Journal of the National Cancer Institute, 82, 4-6 (1989). It hasbeen shown, for example, that tumors which enlarge to greater than 2 mmmust obtain their own blood supply and do so by inducing the growth ofnew capillary blood vessels. Once these new blood vessels becomeembedded in the tumor, they provide a means for tumor cells to enter thecirculation and metastasize to distant sites such as liver, lung or bone(Weidner, N., et al., The New England Journal of Medicine, 324 (1), 1-8(1991).

To date, several naturally occurring angiogenic factors have beendescribed and characterized (Fidler, J. I. and Ellis, L. M., Cell, 79,185-189 (1994). Recently, O'Reilly, et al. have isolated and purified a38 kilodalton (kDa) protein from serum and urine of tumor-bearing micethat inhibits endothelial cell proliferation (O'Reilly, M. et al., Cell,79, 315-328 (1994) and International Application WO 95/29242, publishedNov. 2, 1995. Microsequence analysis of this endothelial inhibitorshowed 98% sequence homology to an internal fragment of murineplasminogen. Angiostatin, as the murine inhibitory fragment was named,was a peptide which included the first four kringle regions of murineplasminogen. A peptide fragment from the same region of humanplasminogen (i.e. containing kringles 1-4) also strongly inhibitedproliferation of capillary endothelial cells in vitro and in vivo. Theintact plasminogen from which this peptide fragment was derived did notpossess as potent an inhibitory effect.

Several angiogenesis inhibitors are currently under development for usein treating angiogenic diseases (Gasparini, G. and Harris, A. L., J.Clin. Oncol., 13 (3): 765-782, (1995), but there are disadvantagesassociated with these compounds. Suramin, for example, is a potentangiogenesis inhibitor but causes severe systemic toxicity in humans atdoses required for antitumor activity. Compounds such as retinoids,interferons and antiestrogens are safe for human use but have weakantiangiogenic effects.

Thus, there is a need for compounds useful in treating angiogenicdiseases in mammals. More specifically, there is a need for angiogenesisinhibitors which are safe for therapeutic use and which exhibitselective toxicity with respect to the pathological condition such as byselectively inhibiting the proliferation of cancer cells whileexhibiting no or a low degree of toxicity to normal (ie. non-cancerous)cells. Such compounds should also be easily and cost-effectively made.

SUMMARY OF THE INVENTION

In its principle embodiment, the present invention provides a kringle 5peptide fragment represented by the structural formula

A-B-C-X-Y (I), or a pharmaceutically acceptible salt thereof, wherein

A is absent or a nitrogen protecting group;

Y is absent or a carboxylic acid protecting group;

B is absent, a naturally-occuring amino acid residue or a peptide ofbetween 2 and 197 amino acids (inclusive), the α-N-terminal optionallycapped with A, the α-C-terminal optionally capped with Y and asubstantial sequence homology to the corresponding amino acid sequencefrom Asp³³⁴ to Arg⁵³⁰ (inclusive) of SEQ ID NO: 1;

C is absent or R¹ -R² -R³ -R⁴ wherein

R¹ is lysyl,

R² is selected from leucyl and arginyl,

R³ is selected from tyrosyl, 3-I-tyrosyl and phenylalanyl and

R⁴ is aspartyl,

with the proviso that at least one of B or C is present, and

X is absent, a naturally occuring amino acid residue or a peptide ofbetween 2 and 11 amino acids (inclusive), the α-N-terminal optionallycapped with A, the α-C-terminal optionally capped with Y and asubstantial sequence homology to the corresponding amino acid sequencebeginning at Tyr⁵³⁵ and ending at Phe⁵⁴⁶ of SEQ ID NO: 1.

The present invention also includes a method for treating a patient inneed of antiangiogenesis therapy comprising adminstering to the patienta compound containing a kringle 5 peptide fragment.

The present invention also includes a composition for treating a patientin need of anti-angiogenesis therapy comprising a compound containing akringle 5 peptide fragment, kringle 5 antisera, kringle 5 receptoragonists and antagonists and kringle 5 antagonists linked to cytotoxicagents either alone or in combination with a pharmaceutically acceptibleexcipient and/or optionally sustained release compounds to form atherapeutic composition.

The present invention also includes a composition for the treatment of adisease selected from the group consisting of cancer, arthritis, maculardegeneration and diabetic retinopathy comprising a compound containing akringle 5 peptide fragment.

The present invention also includes a composition comprising an isolatedsingle or double-stranded polynucleotide sequence that encodes a kringle5 peptide fragment or kringle 5 peptide fragment conjugate. Such apolynucleotide is preferably a DNA molecule. The present invention alsoincludes a vector containing a DNA sequence encoding a kringle 5 peptidefragment or kringle 5 peptide fragment conjugate wherein the vector iscapable of expressing a kringle 5 peptide fragment or kringle 5 peptideconjugate when present in a cell and a composition comprising a cellcontaining a vector wherein the vector contains a DNA sequence encodinga kringle 5 peptide fragment or kringle 5 peptide conjugate. The presentinvention further encompasses gene therapy methods whereby DNA sequencesencoding a kringle 5 peptide fragment or kringle 5 peptide fragmentconjugate are introduced into a patient to modify in vivo kringle 5levels.

The present invention also includes a method of making a kringle 5peptide fragment comprising the steps of: (a) exposing mammalianplasminogen to human or porcine elastase at a ratio of about 1:100 toabout 1:300 to form a mixture of said plasminogen and said elastase; (b)incubating said mixture and (c) isolating the kringle 5 peptide fragmentfrom said mixture.

The present invention also includes a method of making a kringle 5peptide fragment comprising the steps of: (a) exposing mammalianplasminogen to human or porcine elastase at an elastase:plasminogenratio of about 1:100 to about 1:300 to form a mixture of said elastaseand said plasminogen; (b) incubating said mixture; and (c) isolating aprotein conjugate of a kringle 5 peptide fragment from said mixture; (d)exposing said protein conjugate of the kringle 5 peptide fragment topepsin at a ratio of about 1:0.2 to form a mixture of said pepsin andsaid plasminogen and (d) isolating said kringle 5 peptide fragment fromsaid mixture.

The present invention also includes antibodies specific for the kringle5 binding site and methods for the production of antibodies specific forthe kringle 5 binding site. Antibodies can be monoclonal and polyclonaland can be used in diagnostic kits for detection and measurement ofkringle 5 peptide fragment concentrations and for localization ofkringle 5 proteins in tissues and cells. The antibodies specific forkringle 5 can be used in the diagnostic kits to detect the presence andquantity of kringle 5 peptide fragments which are indicative of diseasescaused or exacerbated by angiogenesis, to isolate pure kringle 5 peptidefragments from mixtures containing kringle 5 peptide fragments and toisolate the kringle 5 receptor.

The present invention also includes methods and kits for the detectionand measurement of kringle 5 peptide fragments in body fluid or tissue.The diagnostic kit would be in any configuration well-known to those ofordinary skill in the art and would provide instructions and thenecessary kringle 5 peptide fragments and antisera for the measurementof kringle 5 peptide fragments in biological fluids and tissue extractsof animals and humans with and without tumors.

The present invention also includes a method for preparing kringle 5peptide fragments or prodrugs of kringle 5 peptide fragmentssynthetically by standard methods of solid phase or solution phasechemistry or recombinant technology known to those of ordinary skill inthe art.

The present invention also includes kringle 5 peptide fragments whichcan be labeled isotopically or with other molecules or proteins for usein the detection and visualization of kringle 5 binding sites withtechniques including, but not limited to, radioimmunoassays, competitiveand noncompetitive assays, bioluminescence and enzyme-linkedimmunoabsorbent assays.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of human plasminogen (SEQ ID NO:1).

FIG. 2 shows the comparative homology in amino acid sequences of human(SEQ ID NO: 2), mouse (SEQ ID NO: 8), monkey (SEQ ID NO: 9), bovine (SEQID NO: 10), and porcine (SEQ ID NO: 11) kringle 5.

FIG. 3 shows a graph of the anti-proliferative activity of a single doseof various kringle fragments on bovine capillary endothelial (BCE) cellswhen tested in an in vitro cell proliferation assay.

FIG. 4(a) shows a graph of the anti-proliferative activity of variousconcentrations of kringle 5 (SEQ ID NO: 3) on (BCE) cells when tested inan in vitro cell proliferation assay.

FIG. 4(b) shows a graph of the anti-proliferative activity of variousconcentrations of kringles 1-4 on BCE cells when tested in an in vitrocell proliferation assay.

FIG. 4(c) shows a summary of ED₅₀ values obtained from the inhibition ofvarious kringle fragments on BCE cell proliferation in vitro. In thisFigure: kringle 1 represents the sequence of FIG. 1 from amino acidposition 80 to amino acid position 163, kringle 2.sup.≠ represents thesequence of FIG. 1 from amino acid position 161 to amino acid position245, kringle 3.sup.≠ represents the sequence of FIG. 1 from amino acidposition 253 to amino acid position 335, kringle 4 represents thesequence of FIG. 1 from amino acid position 354 to amino acid position443, kringles 2-3* represents the sequence of FIG. 1 from amino acidposition 161 to amino acid position 335, kringles 1-3 represent thesequence of FIG. 1 from amino acid position 80 to amino acid position353, kringles 1-4 represent the sequence of FIG. 1 from amino acidposition 80 to amino acid position 443, and kringle 5 represents SEQ IDNO: 3.

FIG. 5 shows a graph of the effect of various kringle fragments oninhibition of endothlial cell migration in vitro.

FIGS. 6(a)-6(b) shows the DNA sequence (SEQ ID NO: 12) of humanplasminogen.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "kringle 5" refers to the region of mammalianplasminogen having three disulfide bonds which contribute to thespecific three-dimensional confirmation defined by the fifth kringleregion of the mammalian plasminogen molecule. One such disulfide bondlinks the cysteine residues located at amino acid positions 462 and 541,a second links the cysteine residues located at amino acid positions 483and 524 and a third links the cysteine residues located at amino acidpositions 512 and 536. The amino acid sequence of a complete mammalianplasminogen molecule (the human plasminogen molecule), including itskringle 5 region, is shown in FIG. 1 (SEQ ID NO: 1).

As used herein, the term "kringle 5 peptide fragment" refers to apeptide of between 4 and 104 amino acids (inclusive) with a substantialsequence homology to the corresponding peptide fragment of mammalianplasminogen, an α-N-terminus at about amino acid position 443 of intactmammalian plasminogen and an α-C-terminus at about position 546. Thetotal length of the a kringle 5 peptide fragment may vary depending uponthe manner in which the kringle 5 peptide is obtained or may varysomewhat in sequence depending upon the species from which it isobtained. For example, certain forms of kringle 5 peptide fragments maybe produced by proteolytic cleavage of glu-plasminogen, lys-plasminogenor miniplasminogen using the enzymes human or porcine elastase. Whenproduced in this manner, the α-C-terminal of the peptide resides atabout amino acid 543 of SEQ ID NO: 1, but the α-N-terminal amino acidmay begin at amino acid position 443, 449 or 454. Thus, a kringle 5peptide fragment resulting from human or porcine elastase digestion ofglu-plasminogen, lys-plasminogen or miniplasminogen may have a totallength of either 101 (SEQ ID NO: 2), 95 (SEQ ID NO: 3) or 90 (SEQ ID NO:4) amino acids. A summary of these kringle 5 peptide fragments is shownin Table 1. When produced in the aformentioned manner, a pool of thesethree fragments is obtained wherein about 60% of the fragments have alength of 95 amino acids, about 35% of the fragments have the length of101 amino acids and about 5% of the fragments have a length of 90 aminoacids. If desired, these various fragments may be further purified byreverse phase HPLC, a technique well-known to those skilled in the art.

Alternatively, kringle 5 peptide fragments or kringle 5 peptidefragments bound to protein conjugates may be obtained by expression of arecombinant molecule comprising a polynucleotide having a sequence whichencodes proteins having kringle 5 peptide fragment with an α-N terminalat amino acid position 443, 449 or 454 (preferably at position 443) andan α-C-terminal at amino acid position 543 and 546 (preferably atposition 543) and then purifying the peptide product which is expressed(see Menhart, N., et al, Biochemistry, 32: 8799-8806 (1993). The DNAsequence of human plasminogen has been published (Browne, M. J. et al.Fibrinolysis, 5 (4): 257-260 (1991) and is shown in FIGS. 6(a-b) (SEQ IDNO: 12). A polynucleotide sequence encoding kringle 5 begins at aboutnucleotide position 1421 of SEQ ID NO: 12 and ends at about nucleotideposition 1723. This method of making a kringle 5 peptide fragmentemploys conventional techniques of molecular biology, microbiology,recombinant DNA and immunology, all of which are within the skill of theart and fully explained in the literature. (See for example, "MolecularCloning: A Laboratory Manual" Second Edition by Sambrook et al., ColdSpring Harbor Press, 1989. For example, the gene for a kringle 5 peptidefragment may be isolated from cells or tissues that express high levelsof kringle 5 peptide fragments by (1) isolating messenger RNA from thetissue or cells, (2) using reverse transcriptase to generate thecorresponding DNA sequence and (3) using the polymerase chain reaction(PCR) with the appropriate primers to amplify the DNA sequence codingfor the active kringle 5 amino acid sequence. Furthermore, apolynucleotide encoding a kringle 5 peptide fragment may be cloned intoany commercially available expression vector (such as pBR322, pUCvectors and the like) or expression/purification vectors (such as a GSTfusion vector (Pharmacia, Piscataway, N.J.)) and then expressed in asuitable procaryotic, viral or eucaryotic host. Purification may then beachieved by conventional means or, in the case of a commercialexpression/purification system, in accordance with manufacturer'sinstructions.

Kringle 5 peptide fragments may also be synthesized by standard methodsof solid phase chemistry known to those of ordinary skill in the art Forexample kringle 5 peptide fragments may be synthesized by solid phasechemistry techniques following the procedures described by Steward andYoung (Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis,2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984) using anApplied Biosystem synthesizer. Similarly, multiple fragments may besynthesized then linked together to form larger fragments. Thesesynthetic peptide fragments can also be made with amino acidsubstitutions at specific locations to test for kringle 5 fragment-likeactivity in vitro and in vivo. For solid phase peptide synthesis, asummary of the many techniques may be found in J. M. Stewart and J. D.Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco),1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46,Academic Press (New York), 1973. For classical solution synthesis see G.Schroder and K. Lupke, The Peptides, Vol. 1, Academic Press (New York).In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid is then either attached to an inert solid supportor utilized in solution by adding the next amino acid in the sequencehaving the complimentary (amino or carboxyl) group suitably protectedand under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is added, and soforth. After all the desired amino acids have been linked in the propersequence, any remaining protecting groups (and any solid support) areremoved sequentially or concurrently to afford the final polypeptide. Bysimple modification of this general procedure, it is possible to addmore than one amino acid at a time to a growing chain, for example, bycoupling (under conditions which do not racemize chiral centers) aprotected tripeptide with a properly protected dipeptide to form, afterdeprotection, a pentapeptide.

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis wherein the amino acidα-N-terminal is protected by an acid or base sensitive group. Suchprotecting groups should have the properties of being stable to theconditions of peptide linkage formation while being readily removablewithout destruction of the growing peptide chain or racemization of anyof the chiral centers contained therein. Suitable protecting groups are9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of kringle 5 peptide fragments. Otherpreferred side chain protecting groups are, for side chain amino groupslike lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl). In the solid phasepeptide synthesis method, the α-C-terminal amino acid is attached to asuitable solid support or resin. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. The preferred solidsupport for synthesis of α-C-terminal carboxy peptides is4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene). Thepreferred solid support for α-C-terminal amide peptides is the4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° and 50° C. in a solvent such as dichloromethane or DMF. When thesolid support is4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.The preferred method for coupling to the deprotected 4(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin isis O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer as is well known in the art. In apreferred embodiment, the α-N-terminal in the amino acids of the growingpeptide chain are protected with Fmoc. The removal of the Fmocprotecting group from the α-N-terminal side of the growing peptide isaccomplished by treatment with a secondary amine, preferably piperidine.Each protected amino acid is then introduced in about 3-fold molarexcess, and the coupling is preferably carried out in DMF. The couplingagent is normallyO-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the endof the solid phase synthesis, the polypeptide is removed from the resinand deprotected, either in successively or in a single operation.Removal of the polypeptide and deprotection can be accomplished in asingle operation by treating the resin-bound polypeptide with a cleavagereagent comprising thianisole, water, ethanedithiol and trifluoroaceticacid. In cases wherein the α-C-terminal of the polypeptide is analkylamide, the resin is cleaved by aminolysis with an alkylamine.Alternatively, the peptide may be removed by transesterification, e.g.with methanol, followed by aminolysis or by direct transamidation. Theprotected peptide may be purified at this point or taken to the nextstep directly. The removal of the side chain protecting groups isaccomplished using the cleavage cocktail described above. The fullydeprotected peptide is purified by a sequence of chromatographic stepsemploying any or all of the following types: ion exchange on a weaklybasic resin (acetate form); hydrophobic adsorption chromatography onunderivitized polystyrene-divinylbenzene (for example, Amberlite XAD);silica gel adsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25,LH-20 or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing. Molecular weights ofthese kringle 5 peptide fragments are determined using Fast AtomBombardment (FAB) Mass Spectroscopy. Solid phase kringle 5 peptidefragment synthesis is illustrated in Examples 1 to 12.

Depending on how they are produced, kringle 5 peptide fragments mayexist with or without the aformentioned disulfide bonds of the kringle 5region of mammalian plasminogen or may exist with disulfide bondsforming a tertiary structure which differs from the tertiary structurefound in native mammalian plasminogen (for example, disulfide bondsbetween Cys⁴⁶² and Cys⁴⁸³, between Cys⁵¹² and and between Cys⁵²⁴ andCys⁵³⁶ and Cys⁵⁴¹. Kringle 5 peptide fragments produced by enzymaticcleavage of Glu-, Lys- or miniplasminogen with elastase and/or pepsin(enzymes which cleave at sites removed from the cysteine linkages) willcontain the native tertiary kringle 5 protein structure; kringle 5peptide fragments prepared by solid phase peptide synthesis may or maynot contain cystyl amino acyl residues and kringle 5 peptide fragmentsprepared by expression may contain disulfide bonds at differentpositions than those found in kringle 5 peptide fragments produced byenzymatic cleavage.

As used herein, the term "conjugate of a kringle 5 peptide fragment"means a kringle 5 peptide fragment chemically coupled to another proteinto form a conjugate. Examples of conjugates of kringle 5 peptidefragments include a kringle 5 peptide fragment coupled to albumin or toa peptide fragment from another kringle region of mammalian plasminogen.Molecular weights of conjugates of kringle 5 peptide fragments arebetween about 1,000 and about 25,000 kDa.

As used herein, the term "substantial sequence homology" meansapproximately 60% amino acid identity, desirably at least approximately70% amino acid identity, more desirably approximately 80% amino acididentity and most desirably approximately 95% amino acid identity of thecorresponding peptide sequence of human plasminogen. Because the aminoacid sequence or the number of amino acids in a kringle 5 peptidefragment may vary from species to species or from the method ofproduction, the total number of amino acids in a kringle 5 peptidefragment cannot, in some instances, be defined exactly. Given that thesesequences are identical in at least 73% of their amino acids, it is tobe understood that the amino acid sequence of a kringle 5 peptidefragment is substantially similar among species and that methods ofproduction of kringle 5 peptide fragments provide kringle 5 peptidefragments with substantial sequence homology to the corresponding aminoacid sequences of human plasminogen. FIG. 2 shows the amino acidsequence of a human kringle 5 peptide fragment having 95 amino acids(SEQ ID NO: 2) is in comparison with the sequences of kringle 5fragments from murine (SEQ ID NO: 8), Rhesus monkey (SEQ ID NO: 9),bovine (SEQ ID NO: 10) and porcine (SEQ ID NO: 11) plasminogen.

Thus, the present invention contemplates amino acid residue sequencesthat have substantial sequence homology to the sequences set forthherein such that those sequences demonstrate like biological activity todisclosed kringle 5 peptide fragment sequences. It is well known in theart that modifications and changes can be made without substantiallyaltering the biological function of that peptide. For example,alterations to kringle 5 peptide fragments may enhance the peptide'spotency or stability to enzymatic breakdown. Such contemplated sequencesinclude those analogous sequences characterized by a change in aminoacid residue sequence or type wherein the change does not alter thefundamental nature and biological activity of the aforementioned kringle5 peptide fragments.

A kringle 5 peptide fragment of the present invention may becharacterized on the basis of potency when tested for its ability toinhibit the growth of bovine capillary (BCE) cells in vitro. The data inTable 1 illustrate that the kringle 5 peptide fragment SEQ ID NO: 3 hasa 100-fold increase in activity (i.e. at inhibiting BCE cellproliferation) when compared to the kringle 5 peptide fragment SEQ IDNO: 6 and a 400-fold increase in activity when compared to kringle 14peptide fragments.

As used herein, the term "α-N-terminal" refers to the free alpha-aminogroup of an amino acid in a peptide, and the term "α-C-terminal" refersto the free alpha-carboxylic acid terminus of an amino acid in apeptide.

All peptide sequences are written according to the generally acceptedconvention whereby the α-N-terminal amino acid residue is on the leftand the α-C-terminal is on the right.

As used herein, the term "N-protecting group" refers to those groupsintended to protect the α-N-terminal of an amino acid or peptide or tootherwise protect the amino group of an amino acid or peptide againstundesirable reactions during synthetic procedures. Commonly usedN-protecting groups are disclosed in Greene, "Protective Groups InOrganic Synthesis," (John Wiley & Sons, New York (1981)), which ishereby incorporated by reference. Additionally, protecting groups can beused as prodrugs which are readily cleaved in vivo, for example, byenzymatic hydrolysis, to release the biologically active parent.N-protecting groups comprise loweralkanoyl groups such as formyl, acetyl("Ac"), propionyl, pivaloyl, t-butylacetyl and the like; other acylgroups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups suchas trimethylsilyl and the like. Preferred N-protecting groups areformyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl,benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz). Forexample, lysine may be protected at the α-N-terminal by an acid labilegroup (e.g. Boc) and protected at the ε-N-terminal by a base labilegroup (e.g. Fmoc) then deprotected selectively during synthesis.

As used herein, the term "carboxy protecting group" refers to acarboxylic acid protecting ester or amide group employed to block orprotect the carboxylic acid functionality while the reactions involvingother functional sites of the compound are performed. Carboxy protectinggroups are disclosed in Greene, "Protective Groups in Organic Synthesis"pp. 152-186 (1981), which is hereby incorporated by reference.Additionally, a carboxy protecting group can be used as a prodrugwhereby the carboxy protecting group can be readily cleaved in vivo ,for example by enzymatic hydrolysis, to release the biologically activeparent Such carboxy protecting groups are well known to those skilled inthe art, having been extensively used in the protection of carboxylgroups in the penicillin and cephalosporin fields as described in U.S.Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which are herebyincorporated herein by reference. Representative carboxy protectinggroups are C₁ -C₈ loweralkyl (e.g., methyl, ethyl or t-butyl and thelike); arylalkyl such as phenethyl or benzyl and substituted derivativesthereof such as alkoxybenzyl or nitrobenzyl groups and the like;arylalkenyl such as phenylethenyl and the like; aryl and substitutedderivatives thereof such as 5-indanyl and the like; dialkylaminoalkylsuch as dimethylaminoethyl and the like); alkanoyloxyalkyl groups suchas acetoxymethyl, butyryloxymethyl, valeryloxymethyl,isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl,1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,pivaloyloxymethyl, propionyloxymethyl and the like;cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl,cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl,cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl such asbenzoyloxymethyl, benzoyloxyethyl and the like;arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl,2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl orcycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like;alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and thelike; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)ethyl and the like;alkoxyalkylcarbonyloxyalkyl such as2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl andthe like; arylalkenyloxycarbonyloxyalkyl such as2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like;alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike; alkylaminocarbonylaminoalkyl such asmethylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such asacetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as4-methylpiperazinylcarbonyloxymethyl and the like;dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,diethylaminocarbonylmethyl and the like;(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like; and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.

Representative amide carboxy protecting groups are aminocarbonyl andloweralkylaminocarbonyl groups.

Preferred carboxy-protected compounds of the invention are compoundswherein the protected carboxy group is a loweralkyl, cycloalkyl orarylalkyl ester, for example, methyl ester, ethyl ester, propyl ester,isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amylester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl esterand the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl,aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester. Preferred amidecarboxy protecting groups are loweralkylaminocarbonyl groups. Forexample, aspartic acid may be protected at the α-C-terminal by an acidlabile group (e.g. t-butyl) and protected at the β-C-terminal by ahydrogenation labile group (e.g. benzyl) then deprotected selectivelyduring synthesis.

As used herein, the term "loweralkylaminocarbonyl" means a --C(O)NHR¹⁰group which caps the α-C-terminal of a synthetic, kringle 5 peptidefragment wherein R¹⁰ is C₁ -C₄ alkyl.

As used herein, the term "aminocarbonyl" indicates a --C(O)NH₂ groupwhich caps the α-C-terminal of a synthetic, kringle 5 peptide fragment.

As used herein, the term "prodrug" refers to compounds which are rapidlytransformed in vivo to yield the parent compound, for example, byenzymatic hydrolysis in blood. A thorough discussion is provided in T.Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 ofthe A.C.S. Symposium Series and in Edward B. Roche, ed., BioreversibleCarriers in Drug Design, American Pharmaceutical Association andPermagon Press, 1987.

As used herein, the term "pharmaceutically acceptible prodrug" refers to(1) those prodrugs of the compounds of the present invention which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, commensurate with a suitablebenefit-to-risk ratio and effective for their intended use and (2)zwitterionic forms, where possible, of the parent compound.

As used herein, the term "antiangiogenesis activity" refers to thecapability of a molecule to inhibit the growth of blood vessels.

As used herein, the term "endothelial inhibiting activity" refers to thecapability of a molecule to inhibit angiogenesis in general and, forexample, to inhibit the growth or migration of bovine capillaryendothelial cells in culture in the presence of fibroblast growth factoror other known growth factors.

As used herein, the term "ED₅₀ " is an abbreviation for the dose of akringle 5 peptide fragment which is effective to inhibit the growth ofblood vessels or inhibit the growth of bovine capillary endothelialcells in culture in the presence of fibroblast growth factor or otherknown growth factors or inhibit the migration of endeothelial cells byone-half of what the growth or migration would be in the absence of theinhibitor.

As used herein, for the most part, the names of naturally-occuring aminoacids and aminoacyl residues used herein follow the naming conventionssuggested by the IUPAC Commission on the Nomenclature of OrganicChemistry and the IUPAC-IUB Commission on Biochemical Nomenclature asset out in Nomenclature of α-Amino Acids (Recommendations, 1974),Biochemistry, 14 (2), (1975). Accordingly, the terms "Ala," "Arg,""Asn," "Asp," "Cys," "Gln," "Glu," "Gly," "His," "Ile," "Leu," "Lys,""Met," "Phe," "Pro," "Ser," "Thr," "Trp," "Tyr" and "Val" refer to theamino acids alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine and their corresponding aminoacylresidues in peptides in their L-, D- or D, L- forms. Where no specificconfiguration is indicated, one skilled in the art would understand thatthe stereochemistry of the α-carbon of the amino acids and aminoacylresidues in peptides described in this specification and the appendedclaims is the naturally occuring or "L" configuration with the exceptionof the achiral molecule glycine and with the further exception of anyamino acids which are achiral or otherwise designated as "D-."

As used herein, the term "3-I-Tyr" means a L-, D-, or D, L-tyrosylresidue wherein a hydrogen radical ortho to the phenolic hydroxyl isreplaced by an iodide radical. The iodide radical may be radioactive ornonradioactive.

The present invention also contemplates amino acid residues withnon-naturally occuring side chain residues such as homophenylalanine,phenylglycine, norvaline, norleucine, ornithine, thiazoylalanine (2-, 4-and 5- substituted) and the like.

Thus, it is to be understood that the present invention is contemplatedto encompass any derivatives of kringle 5 peptide fragments which haveantiangiogenic activity and includes the entire class of kringle 5peptide fragments described herein and derivatives of those kringle 5peptide fragments. Additionally, the invention is not dependent on themanner in which the kringle 5 peptide fragment is produced, i.e. by (1)proteolytic cleavage of an isolated mammalian plasminogen, (2) byexpression of a recombinant molecule having a polynucleotide whichencodes the amino acid sequence of a kringle 5 peptide fragment or aconjugate containing a kringle 5 peptide fragment and (3) solid phasesynthetic techniques known to those of ordinary skill in the art.

In one embodiment, the present invention provides peptides with thegeneral structure B-C-X wherein B is a 88-mer peptide beginning atVal⁴⁴³ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined, R² is leucyl; R³ is tyrosyl and X is a 9-mer peptide beginningat Tyr⁵³⁵ and ending at Ala⁵⁴³ of SEQ ID NO: 1 (SEQ ID NO: 2).

In another embodiment, the present invention provides peptides with thegeneral structure B-C-X wherein B is a 82-mer peptide beginning atVal⁴⁴⁹ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined; R² is leucyl; R³ is tyrosyl and X is a 9-mer peptide beginningat Tyr⁵³⁵ and ending at Ala⁵⁴³ of SEQ ID NO: 1 (SEQ ID NO: 3).

In yet another embodiment, the present invention provides peptides withthe general structure B-C-X wherein B is a 77-mer peptide beginning atVal⁴⁵⁴ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined; R² is leucyl; R³ is tyrosyl and X is a 9-mer peptide beginningat Tyr⁵³⁵ and ending at Ala⁵⁴³ of SEQ ID NO: 1 (SEQ ID NO: 4).

In yet another embodiment, the present invention provides peptides withthe general structure B-C-X wherein B is a 88-mer peptide beginning atVal⁴⁴³ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined; R² is leucyl; R³ is tyrosyl and X is a 12-mer peptide beginningat Tyr⁵³⁵ and ending at Phe⁵⁴⁶ of SEQ ID NO: 1 (SEQ ID NO: 5).

In yet another embodiment, the present invention provides peptides withthe general structure structure B-C-X wherein B is a 82-mer peptidebeginning at Val⁴⁴⁹ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴are previously defined; R² is leucyl; R³ is tyrosyl and X is a 12-merpeptide beginning at Tyr⁵³⁵ and ending at Phe⁵⁴⁶ of SEQ ID NO: 1 (SEQ IDNO: 6).

In yet another embodiment, the present invention provides peptides withthe general structure B-C-X wherein B is a 77-mer peptide beginning atVal⁴⁵⁴ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined; R² is leucyl, R³ is tyrosyl and X is a 12-mer peptide beginningat Tyr⁵³⁵ and ending at Phe⁵⁴⁶ of SEQ ID NO: 1 (SEQ ID NO: 7).

In yet another embodiment, the present invention provides peptides withthe general structure B-C-X wherein B is a 176-mer peptide beginning atVal³⁵⁵ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined; R² is leucyl, R³ is tyrosyl and X is a 12-mer peptide beginningat Tyr⁵³⁵ and ending at Ala⁵⁴³ of SEQ ID NO: 1.

In yet another embodiment, the present invention provides peptides withthe general structure B-C-X wherein B is a 176-mer peptide beginning atVal³⁵⁵ and ending at Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previouslydefined; R² is leucyl, R³ is tyrosyl and X is a 12-mer peptide beginningat Tyr⁵³⁵ and ending at Phe⁵⁴⁶ of SEQ ID NO: 1.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-Y wherein A is acetyl, B is a 13-mer peptidebeginning at amino acid Val⁴⁴⁹ and ending at Asp⁴⁶¹ of SEQ ID NO: 1 andY is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-Y wherein A is acetyl, B is a 20-mer peptidebeginning at amino acid Met⁴⁶³ and ending at Pro⁴⁸² of SEQ ID NO: 1 andY is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-Y wherein A is acetyl, B is a 28-mer peptidebeginning at amino acid Gln⁴⁸⁴ and ending at amino acid Tyr⁵¹¹ of SEQ IDNO: 1 and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-Y wherein A is acetyl; B is an 11-mer peptidebeginning at amino acid position Arg⁵¹³ and ending at amino acidposition Trp⁵²³ of SEQ ID NO: 1 and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-C-Y wherein A is acetyl; B is a dipeptidebeginning at amino acid position Pro⁵²⁹ and ending at amino acidposition Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previously defined; R²is leucyl; R³ is tyrosyl and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-C-Y wherein A is acetyl; B is a dipeptidebeginning at amino acid position Pro⁵²⁹ and ending at amino acidposition Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previously defined; R²is leucyl; R³ is tyrosyl and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-C-X-Y wherein A is acetyl; B is a hexapeptidebeginning at amino acid position Tyr⁵²⁵ and ending at amino acidposition Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previously defined; R²is leucyl, R³ is tyrosyl, X is tyrosyl and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-C-X-Y wherein A is acetyl; B is arginyl; C, R¹and R⁴ are previously defined; R² is leucyl; R³ is tyrosyl; X is tyrosyland Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-C-X-Y wherein A is acetyl, B is a dipeptidebeginning at amino acid position Pro⁵²⁹ and ending at amino acidposition Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previously defined; R²is leucyl; R³ is tyrosyl; X is 3-I-tyrosyl and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-B-C-X-Y wherein A is acetyl, B is a dipeptidebeginning at amino acid position Pro⁵²⁹ and ending at amino acidposition Arg⁵³⁰ of SEQ ID NO: 1; C, R¹ and R⁴ are previously defined; R²is leucyl; R³ is 3-I-tyrosyl; X is tyrosyl and Y is aminocarbonyl.

In yet another embodiment, the present invention provides peptides withthe general structure A-C-Y wherein A is acetyl; C, R¹ and R⁴ arepreviously defined; R² is leucyl, R³ is tyrosyl, X is tyrosyl and Y isaminocarbonyl.

Representative compounds of formula (I) include:

H₂ N-Val⁴⁴³ -Ala⁵⁴³ -CO₂ H of SEQ ID NO: 1 (SEQ ID NO: 2)

H₂ N-Val⁴⁴⁹ -Ala⁵⁴³ -CO₂ H of SEQ ID NO: 1 (SEQ ID NO: 3)

H₂ N-Val⁴⁵⁴ -Ala⁵⁴³ -CO₂ H of SEQ ID NO: 1 (SEQ ID NO: 4)

H₂ N-Val⁴⁴³ -Phe⁵⁴⁶ -CO₂ H of SEQ ID NO: 1 (SEQ ID NO: 5)

H₂ N-Val⁴⁴⁹ -Phe⁵⁴⁶ -CO₂ H of SEQ ID NO: 1 (SEQ ID NO: 6)

H₂ N-Val⁴⁵⁴ -Phe⁵⁴⁶ -CO₂ H of SEQ ID NO: 1 (SEQ ID NO: 7)

N-Ac-Val⁴⁴⁹ -Asp⁴⁶¹ -NH₂ of SEQ ID NO: 1

N-Ac-Met⁴⁶³ -Pro⁴⁸² -NH₂ of SEQ ID NO: 1

N-Ac-Gln⁴⁸⁴ -Tyr⁵¹¹ -NH₂ of SEQ ID NO: 1

N-Ac-Arg⁵¹³ -Trp⁵²³ -NH₂ of SEQ ID NO: 1

N-Ac-Tyr⁵²⁵ -Tyr⁵³⁵ -NH₂ of SEQ ID NO: 1

N-Ac-Pro⁵²⁹ -Tyr⁵³⁵ -NH₂ of SEQ ID NO: 1

N-Ac-Pro⁵²⁹ -Asp⁵³⁴ -NH₂ of SEQ ID NO: 1

N-Ac-Arg⁵³⁰ -Tyr⁵³⁵ -NH₂ of SEQ ID NO: 1

N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH₂ (SEQ ID NO: 13)

N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH₂ (SEQ ID NO: 14)

N-Ac-H-Lys⁵³¹ -Asp⁵³⁴ -NH₂ of SEQ ID NO: 1

H₂ N-Val³⁵⁵ -Ala⁵⁴³ -CO₂ H of SEQ ID NO: 1

H₂ N-Val³⁵⁵ -Phe⁵⁴⁶ -CO₂ H of SEQ ID NO: 1

The compounds of the invention, including but not limited to thosespecified in the examples, possess anti-angiogenic activity. Asangiogenesis inhibitors, such compounds are useful in the treatment ofboth primary and metastatic solid tumors and carcinomas of the breast;colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach;pancreas; liver; gallbladder; bile ducts; small intestine; urinary tractincluding kidney, bladder and urothelium; female genital tract includingcervix, uterus, ovaries, choriocarcinoma and gestational trophoblasticdisease; male genital tract including prostate, seminal vesicles, testesand germ cell tumors; endocrine glands including thyroid, adrenal, andpituitary; skin including hemangiomas, melanomas, sarcomas arising frombone or soft tissues and Kaposi's sarcoma; tumors of the brain, nerves,eyes, and meninges including astrocytomas, gliomas, glioblastomas,retinoblastomas, neuromas, neuroblastomas, Schwannomas and meningiomas;solid tumors arising from hematopoietic malignancies such as leukemiasand including chloromas, plasmacytomas, plaques and tumors of mycosisfungoides and cutaneous T-cell lymphoma/leukemia; lymphomas includingboth Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of autoimmunediseases including rheumatoid, immune and degenerative arthritis; oculardiseases including diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, retrolental fibroplasia, neovascular glaucoma,rubeosis, retinal neovascularization due to macular degeneration andhypoxia; abnormal neovascularization conditions of the eye; skindiseases including psoriasis; blood vessel diseases including hemagiomasand capillary proliferation within atherosclerotic plaques; Osler-WebberSyndrome; myocardial angiogenesis; plaque neovascularization;telangiectasia; hemophiliac joints; angiofibroma; wound granulation;diseases characterized by excessive or abnormal stimulation ofendothelial cells including intestinal adhesions, Crohn's disease,atherosclerosis, scleroderma and hypertrophic scars (i.e. keloids) anddiseases which have angiogenesis as a pathologic consequence includingcat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacterpylori). Another use is as a birth control agent which inhibitsovulation and establishment of the placenta.

The compounds of the present invention may also be useful for theprevention of metastases from the tumors described above either whenused alone or in combination with radiotherapy and/or otherchemotherapeutic treatments conventionally administered to patients fortreating angiogenic diseases. For example, when used in the treatment ofsolid tumors, compounds of the present invention may be administeredwith chemotherapeutic agents such as alpha inteferon, COMP(cyclophosphamide, vincristine, methotrexate and prednisone), etoposide,mBACOD (methortrexate, bleomycin, doxorubicin, cyclophosphamide,vincristine and dexamethasone), PROMACE/MOPP (prednisone, methotrexate(w/leucovin rescue), doxorubicin, cyclophosphamide, taxol,etoposide/mechlorethamine, vincristine, prednisone and procarbazine),vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan,thalidomide, SP-PG and the like. Other chemotherapeutic agents includealkylating agents such as nitrogen mustards including mechloethamine,melphan, chlorambucil, cyclophosphamide and ifosfamide; nitrosoureasincluding carmustine, lomustine, semustine and streptozocin; alkylsulfonates including busulfan; triazines including dacarbazine;ethyenimines including thiotepa and hexamethylmelamine; folic acidanalogs including methotrexate; pyrimidine analogues including5-fluorouracil, cytosine arabinoside; purine analogs including6-mercaptopurine and 6-thioguanine; antitumor antibiotics includingactinomycin D; the anthracyclines including doxorubicin, bleomycin,mitomycin C and methramycin; hormones and hormone antagonists includingtamoxifen and cortiosteroids and miscellaneous agents includingcisplatin and brequinar. For example, a tumor may be treatedconventionally with surgery, radiation or chemotherapy and kringle 5administration with subsequent kringle 5 adminsteration to extend thedormancy of micrometastases and to stabilize and inhibit the growth ofany residual primary tumor.

Cytotoxic agents such as ricin may be linked to kringle 5 peptidefragments and thereby provide a tool for destruction of cells that bindkringle 5. Peptides linked to cytotoxic agents may be infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity kringle 5 fragments may be deliveredvia cannula directly into the target or into vessels supplying thetarget site. Such agents may also be delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae. A combination ofkringle 5 antagonists may be co-applied with stimulators of angiogenesisto increase vascularization of tissue. Therapeutic regimens of this typecould provide an effective means of destroying metastatic cancer.

The compounds of the present invention may be used in the form ofpharmaceutically acceptible salts derived from inorganic or organicacids. By "pharmaceutically acceptible salt" is meant those salts whichare, within the scope of sound medical judgement, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptible salts are well-known in the art. For example, S. M. Berge, etal. describe pharmaceutically acceptible salts in detail in J.Pharmaceutical Sciences, 1977, 66: 1 et seq., which is herebyincorporated herein by reference. The salts may be prepared in situduring the final isolation and purification of the compounds of theinvention or separately by reacting a free base function with a suitableorganic acid. Representative acid addition salts include, but are notlimited to acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups can be quarternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid.

Basic addition salts can be prepared in situ during the final isolationand purification of kringle 5 peptide fragments by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptible metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptible salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic anines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Preferred salts of the compounds ofthe invention include phosphate, tris and acetate.

Kringle 5 peptide fragments, kringle 5 antisera, kringle 5 receptoragonists, kringle 5 receptor antagonists or combinations thereof may becombined with pharmaceutically acceptible sustained-release matrices,such as biodegradable polymers, to form therapeutic compositions. Asustained-release matrix, as used herein, is a matrix made of materials,usually polymers, which are degradable by enzymatic or acid-basehydrolysis or by dissolution. Once inserted into the body, the matrix isacted upon by enzymes and body fluids. A sustained-release matrix isdesirably chosen from biocompatible materials such as liposomes,polylactides (polylactic acid), polyglycolide (polymer of glycolicacid), polylactide co-glycolide (copolymers of lactic acid and glycolicacid) polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

Kringle 5 peptide fragments, kringle 5 receptor agonists, kringle 5receptor antagonists or combinations thereof may be combined withpharmaceutically acceptable excipients or carriers to form therapeuticcompositions. A pharmaceutically acceptable carrier or excipient refersto a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thecompositions may be administered parenterally, sublingually,intracisternally, intravaginally, intraperitoneally, rectally, bucallyor topically (as by powder, ointment, drops, transdermal patch oriontophoresis device).

The term "parenteral," as used herein, refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. Pharmaceuticalcompositions for parenteral injection comprise pharmaceuticallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol and the like), carboxymethylcellulose andsuitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity may bemaintained, for example, by the use of coating materials such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants. These compositions mayalso contain adjuvants such as preservatives, wetting agents,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents such as paraben, chlorobutanol, phenol sorbic acidand the like. It may also be desirable to include isotonic agents suchas sugars, sodium chloride and the like. Prolonged absorption of theinjectable pharmaceutical form may be brought about by the inclusion ofagents, such as aluminum monostearate and gelatin, which delayabsorption. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use.

Topical administration includes administration to the skin, mucosa andsurfaces of the lung and eye. Compositions for topical administration,including those for inhalation, may be prepared as a dry powder whichmay be pressurized or non-pressurized. In non-pressurized powdercompositions, the active ingredient in finely divided form may be usedin admixture with a larger-sized pharmaceutically acceptable inertcarrier comprising particles having a size, for example, of up to 100micrometers in diameter. Suitable inert carriers include sugars such aslactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 micrometers. For topical administration to the eye, a compound ofthe invention is delivered in a pharmaceutically acceptable ophthalmicvehicle such that the compound is maintained in contact with the ocularsurface for a sufficient time period to allow the compound to penetratethe corneal and internal regions of the eye, as, for example, theanterior chamber, posterior chamber, vitreous body, aqueous humor,vitreous humor, cornea, iris/cilary, lens, choroid/retina and sclera.The pharmaceutically acceptable ophthalmic vehicle may, for example, bean ointment, vegetable oil or an encapsulating material. Alternatively,a compound of the invention may be injected directly into the vitriousand aqueous humor.

The composition may be pressurized and contain a compressed gas such asnitrogen or a liquified gas propellant. The liquified propellant mediumand indeed the total composition is preferably such that the activeingredient does not dissolve therein to any substantial extent. Thepressurized composition may also contain a surface active agent such asa liquid or solid non-ionic surface active agent or may be a solidanionic surface active agent. It is preferred to use the solid anionicsurface active agent in the form of a sodium salt.

Compositions for rectal or vaginal administration are preferablysuppositories which may be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidsat room temperature but liquids at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Compounds of the present invention may also be administered in the formof liposomes. As is known in the art, liposomes are generally derivedfrom phospholipids or other lipid substances. Liposomes are formed bymono- or multi-lamellar hydrated liquid crystals that are dispersed inan aqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form may contain, in addition to acompound of the present invention, stabilizers, preservatives,excipients and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33 et seq., which is hereby incorporated herein byreference.

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds of the present invention may be employedin pure form or, where such forms exist, in pharmaceutically acceptablesalt form and with or without a pharmaceutically acceptible excipient. A"therapeutically effective amount" of the compound of the inventionmeans a sufficient amount of the compound to treat an angiogenic disease(for example, to limit tumor growth or to slow or block tumormetastasis) at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidential with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.Total daily dose of kringle 5 peptide fragments to be administeredlocally or systemically to a human or other mammal host in single ordivided doses may be in amounts, for example, from 0.0001 to 200 mg/kgbody weight daily and more usually 1 to 300 mg/kg body weight. Ifdesired, the effective daily dose may be divided into multiple doses forpurposes of administration. Consequently, single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.

It will be understood that agents which can be combined with thecompound of the present invention for the inhibition, treatment orprophylaxis of angiogenic diseases are not limited to those listedabove, but include, in principle, any agents useful for the treatment orprophylaxis of angiogenic diseases.

The present invention also encompasses gene therapy whereby the geneencoding kringle 5 peptide fragments or kringle 5 peptide fragmentconjugates is regulated in a patient. Various methods of transferring ordelivering DNA to cells for expression of the gene product protein,otherwise referred to as gene therapy, are disclosed in Gene Transferinto Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev. Biotechn. 12(4): 335-356 (1992), which is hereby incorporated herein by reference.Gene therapy encompasses incorporation of DNA sequences into somaticcells or germ line cells for use in either ex vivo or in vivo therapy.Gene therapy functions to replace genes, to augment normal or abnormalgene function and to combat infectious diseases and other pathologies.

Strategies for treating medical problems with gene therapy includetherapeutic strategies such as identifying the defective gene and thenadding a functional gene to either replace the function of the defectivegene or to augment a slightly functional gene or prophylactic strategiessuch as adding a gene which encodes a protein product that will treatthe condition or that will make the tissue or organ more susceptible toa treatment regimen. As an example of a prophylactic strategy, a geneencoding a kringle 5 peptide fragment or a kringle 5 peptide fragmentconjugate may be placed in a patient and thus prevent occurrence ofangiogenesis or a gene that makes tumor cells more susceptible toradiation could be inserted so that radiation of the tumor would causeincreased killing of the tumor cells.

Many protocols for the transfer of kringle 5 peptide fragment DNA orkringle 5 peptide fragment regulatory sequences are envisioned in thisinvention. Transfection of promoter sequences, other than onesspecifically associated with a kringle 5 peptide fragment or othersequences which would increase production of kringle 5 peptidefragments, are also envisioned as methods of gene therapy. An example ofthis technology is found in Transkaryotic Therapies, Inc., of Cambridge,Mass., using homologous recombination to insert a "genetic switch" whichturns on an erythropoietin gene in cells as disclosed in GeneticEngineering News, Apr. 15, 1994, which is hereby incorporated herein byreference. Such "genetic switches" could be used to activate a kringle 5peptide fragment (or a kringle 5 receptor) in cells not normallyexpressing these proteins.

Gene transfer methods for gene therapy fall into three broad categories:(1) physical (e.g., electroporation, direct gene transfer and particlebombardment), (2) chemical (e.g. lipid-based carriers and othernon-viral vectors) and (3) biological (e.g. virus derived vectors). Forexample, non-viral vectors such as liposomes coated with DNA may bedirectly injected intravenously into the patient It is believed that theliposome/DNA complexes are concentrated in the liver where they deliverthe DNA to macrophages and Kupffer cells. Vectors or the "naked" DNA ofthe gene may also be directly injected into the desired organ, tissue ortumor for targeted delivery of the therapeutic DNA.

Gene therapy methodologies can also be described by delivery site.Fundamental ways to deliver genes include ex vivo gene transfer, in vivogene transfer and in vitro gene transfer. In ex vivo gene transfer,cells are taken from the patient and grown in cell culture. The DNA istransfected into the cells, and the transfected cells are expanded innumber and then reimplanted in the patient. In in vitro gene transfer,the transformed cells are cells growing in culture, such as tissueculture cells, and not particular cells from a particular patient. These"laboratory cells" are transfected, and the transfected cells areselected and expanded for either implantation into a patient or forother uses. In vivo gene transfer involves introducing the DNA into thecells of the patient when the cells are within the patient All three ofthe broad based categories described above may be used to achieve genetransfer in vivo, ex vivo and in vitro.

Mechanical (i.e. physical) methods of DNA delivery can be achieved bymicroinjection of DNA into germ or somatic cells, pneumaticallydelivered DNA-coated particles such as the gold particles used in a"gene gun" and inorganic chemical approaches such as calcium phosphatetransfection. It has been found that physical injection of plasmid DNAinto muscle cells yields a high percentage of cells which aretransfected and have a sustained expression of marker genes. The plasmidDNA may or may not integrate into the genome of the cells.Non-integration of the transfected DNA would allow the transfection andexpression of gene product proteins in terminally differentiated,non-proliferative tissues for a prolonged period of time without fear ofmutational insertions, deletions or alterations in the cellular ormitochondrial genome. Long-term, but not necessarily permanent, transferof therapeutic genes into specific cells may provide treatments forgenetic diseases or for prophylactic use. The DNA could be reinjectedperiodically to maintain the gene product level without mutationsoccurring in the genomes of the recipient cells. Non-integration ofexogenous DNAs may allow for the presence of several different exogenousDNA constructs within one cell with all of the constructs expressingvarious gene products.

Particle-mediated gene transfer may also be employed for injecting DNAinto cells, tissues and organs. With a particle bombardment device, or"gene gun," a motive force is generated to accelerate DNA-coated highdensity particles (such as gold or tungsten) to a high velocity thatallows penetration of the target organs, tissues or cells.Electroporation for gene transfer uses an electrical current to makecells or tissues susceptible to electroporation-mediated gene transfer.A brief electric impulse with a given field strength is used to increasethe permeability of a membrane in such a way that DNA molecules canpenetrate into the cells. The techniques of particle-mediated genetransfer and electroporation are well known to those of ordinary skillin the art

Chemical methods of gene therapy involve carrier-mediated gene transferthrough the use of fusogenic lipid vesicles such as liposomes or othervesicles for membrane fusion. A carrier harboring a DNA of interest canbe conveniently introduced into body fluids or the bloodstream and thensite specifically directed to the target organ or tissue in the body.Cell or organ-specific DNA-carrying liposomes, for example, can bedeveloped and the foreign DNA carried by the liposome absorbed by thosespecific cells. Injection of immunoliposomes that are targeted to aspecific receptor on certain cells can be used as a convenient method ofinserting the DNA into the cells bearing that receptor. Another carriersystem that has been used is the asialoglycoprotein/polylysine conjugatesystem for carrying DNA to hepatocytes for in vivo gene transfer.

Transfected DNA may also be complexed with other kinds of carriers sothat the DNA is carried to the recipient cell and then deposited in thecytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclearproteins in specifically engineered vesicle complexes and carrieddirectly into the nucleus.

Carrier mediated gene transfer may also involve the use of lipid-basedcompounds which are not liposomes. For example, lipofectins andcytofectins are lipid-based positive ions that bind to negativelycharged DNA and form a complex that can ferry the DNA across a cellmembrane. Another method of carrier mediated gene transfer involvesreceptor-based endocytosis. In this method, a ligand (specific to a cellsurface receptor) is made to form a complex with a gene of interest andthen injected into the bloodstream. Target cells that have the cellsurface receptor will specifically bind the ligand and transport theligand-DNA complex into the cell.

Biological gene therapy methodologies employ viral vectors to insertgenes into cells. The term "vector" as used herein means a carrier whichmay contain or associate with specific polynucleotide sequences andwhich functions to transport the specific polynucleotide sequences intoa cell. The transfected cells may be cells derived from the patient'snormal tissue, the patient's diseased tissue or non-patient cells.Examples of vectors include plasmids and infective microorganisms suchas viruses or nonviral vectors such as the ligand-DNA conjugates,liposomes and the lipid-DNA complexes discussed above.

It may be desirable that a recombinant DNA molecule comprising a kringle5 peptide fragment DNA sequence is operatively linked to an expressioncontrol sequence to form an expression vector capable of expressing akringle 5 peptide fragment. Alternatively, gene regulation of kringle 5peptide fragments may be accomplished by administering compounds thatbind to the kringle 5 gene or control regions associated with thekringle 5 gene or its corresponding RNA transcript to modify the rate oftranscription or translation.

Viral vectors that have been used for gene therapy protocols include,but are not limited to, retroviruses, other RNA viruses such aspoliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpesviruses, SV 40, vaccinia and other DNA viruses. Replication-defectivemurine retroviral vectors are the most widely utilized gene transfervectors. Murine leukemia retroviruses are composed of a single strandRNA completed with a nuclear core protein and polymerase (pol) enzymesencased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include gag, pol, and env genes enclosed at the 5' and 3'long terminal repeats (LTRs). Retroviral vector systems exploit the factthat a minimal vector containing the 5' and 3' LTRs and the packagingsignal are sufficient to allow vector packaging and infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line. Fundamentaladvantages of retroviral vectors for gene transfer include efficientinfection and gene expression in most cell types, precise single copyvector integration into target cell chromosomal DNA and ease ofmanipulation of the retroviral genome. For example, altered retrovirusvectors have been used in ex vivo methods to introduce genes intoperipheral and tumor-infiltrating lymphocytes, hepatocytes, epidermalcells, myocytes or other somatic cells (which may then be introducedinto the patient to provide the gene product from the inserted DNA).

The adenovirus is composed of linear, double stranded DNA complexed withcore proteins and surrounded with capsid proteins. Advances in molecularvirology have led to the ability to exploit the biology of theseorganisms to create vectors capable of transducing novel geneticsequences into target cells in vivo. Adenoviral-based vectors willexpress gene product peptides at high levels. Adenoviral vectors havehigh efficiencies of infectivity, even with low titers of virus.Additionally, the virus is fully infective as a cell-free virion soinjection of producer cell lines are not necessary. Another potentialadvantage to adenoviral vectors is the ability to achieve long termexpression of heterologous genes in vivo.

Viral vectors have also been used to insert genes into cells using invivo protocols. To direct tissue-specific expression of foreign genes,cis-acting regulatory elements or promoters that are known to betissue-specific may be used. Alternatively, this can be achieved usingin situ delivery of DNA or viral vectors to specific anatomical sites invivo. For example, gene transfer to blood vessels in vivo was achievedby implanting in vitro transduced endothelial cells in chosen sites onarterial walls. The virus-infected surrounding cells, in turn, alsoexpressed the gene product. A viral vector can be delivered directly tothe in vivo site (by catheter, for example) thus allowing only certainareas to be infected by the virus and providing long-term, site-specificgene expression. In vivo gene transfer using retrovirus vectors has alsobeen demonstrated in mammary tissue and hepatic tissue by injection ofthe altered virus into blood vessels leading to the organs.

Kringle 5 peptide fragments may also be produced and used in a varietyof applications. As examples, different peptide fragments of kringle 5can be used (1) as agonists and antagonists active at kringle 5 bindingsites, (2) as antigens for the development of specific antisera, (3) aspeptides for use in diagnostic kits and (4) as peptides linked to orused in combination with cytotoxic agents for targeted killing of cellsthat bind kringle 5 peptide fragments. The amino acid sequences thatcomprise these peptide fragments may be selected on the basis of theirposition on the exterior regions of the molecule which are accessiblefor binding to antisera or the inhibitory potency of the peptidefragments toward processes arising from or exaserbated by angiogenesis.Furthermore, these peptide sequences may be compared to known sequencesusing protein sequence databases such as GenBank, Brookhaven Protein,SWISS-PROT, and PIR to determine potential sequence homologies. Thisinformation facilitates elimination of sequences that exhibit a highdegree of sequence homology to other molecules and thereby enhances thepotential for high specificity in the development of antisera, agonistsand antagonists to kringle 5.

Kringle 5 peptide fragments may also be used as a means to isolate akringle 5 receptor by immobilization of the kringle 5 peptide fragmenton a solid support in, for example, an affinity column through whichcultured endothelial cells or membrane extracts are passed. As is knownin the art, isolation and purification of a kringle 5 receptor may befollowed by amino acid sequencing to identify and isolatepolynucleotides which encode the kringle 5 receptor. Suchpolynucleotides may then be cloned into a suitable expression vector andtransfected into tumor cells. Expression of the receptor by thetransfected tumor cells would enhance the responsiveness of these cellsto endogenous or exogenous kringle 5 peptide fragments and therebydecrease the rate of metastatic growth. Furthermore, recombinantexpression of this receptor would allow greater amounts of receptor tobe produced, e.g. to produce a sufficient quantity for use in highthroughput screening assays to identify smaller antagonists which mimicthe action of kringle 5.

Systematic substitution of amino acids within these synthesized peptidesmay yield high affinity peptide agonists and antagonists to the kringle5 receptor that enhance or diminish kringle 5 peptide fragment bindingto its receptor. Such agonists may be used to suppress the growth ofmicrometastases and thereby limit the spread of cancer. In cases ofinadequate vascularization, antagonists to kringle 5 peptide fragmentsmay be applied to block the inhibitory effects of kringle 5 peptidefragments and promote angiogenesis. For example, this type of treatmentmay have therapeutic effects in promoting wound healing in diabetics.

Kringle 5 peptide fragments of the present invention can also be used asantigens to generate polyclonal or monoclonal antibodies which arespecific for the kringle 5 inhibitor. One way in which such antibodiescould be used is in diagnostic methods and kits to detect or quantifykringle 5 peptide fragments in a body fluid or tissue. Results fromthese tests could be used to diagnose or determine the prognosticrelevance of kringle 5 peptide fragments.

Kringle 5 peptide fragments may be labeled with radioactive isotopes(See Example 13) or chemically coupled to proteins to form conjugates.Conjugates include enzymes, carrier proteins, cytotoxic agents,fluorescent, chemiluminescent and bioluminescent molecules which areused to facilitate the testing of the ability of compounds containingkringle 5 peptide fragments to bind kringle 5 antisera, detect celltypes which possess a kringle 5 peptide fragment receptor or aid inpurification of kringle 5 peptide fragments. The coupling technique isgenerally chosen on the basis of the functional groups available on theamino acids of the kringle 5 peptide fragment sequence including, butnot limited to alkyl, amino, sulfhydryl, carboxyl, amide, phenol,indolyl and imidazoyl. Various reagents used to effect such couplingsinclude, among others, glutaraldehyde, diazotized benzidine,carbodiimides and p-benzoquinone. The efficiency of the couplingreaction is determined using different techniques appropriate for thespecific reaction. For example, radiolabeling of a kringle 5 peptide ora biologically active fragment thereof with I¹²⁵ may be accomplishedusing chloramine T and NaI¹²⁵ of high specific activity. The reaction isterminated with sodium metabisulfite and the mixture is desalted ondisposable columns. The labeled peptide is eluted from the column andthe fractions are collected. Aliquots are removed from each fraction andradioactivity is measured in a gamma counter. This procedure providesthe radiolabeled kringle 5 peptide fragment free from unreacted NaI¹²⁵.In another example, blood or tissue extracts containing a kringle 5peptide fragment coupled to kringle 4 may be purified on a polylysineresin affinity column whereby the kringle 4-kringle 5 peptide fragmentbinds to the resin through the affinity of the kringle 4 peptidefragment for lysine. Elution of the bound protein would provide apurified kringle 4-kringle 5 peptide fragment

Another application of peptide conjugation is the production ofpolyclonal antisera. The production of antiserum against kringle 5peptide fragments, kringle 5 peptide fragment analogs and the kringle 5receptor can be performed using established techniques known to thoseskilled in the art. For example, kringle 5 peptide fragments containinglysine residues may be linked to purified bovine serum albumin (BSA)using glutaraldehyde. The efficiency of this reaction may be determinedby measuring the incorporation of radiolabeled peptide. Unreactedglutaraldehyde and peptide may be separated by dialysis, and theconjugate may be use to raise polyclonal antisera in rabbits, sheep,goats or other animals. Kringle 5 peptide fragments conjugated to acarrier molecule such as BSA may be combined with an adjuvant mixture,emulsified and injected subcutaneously at multiple sites on the back,neck, flanks, and sometimes in the footpads of a suitable host.Generally, booster injections are then given at regular intervals, suchas every 2 to 4 weeks. Approximately 7 to 10 days after each injection,blood samples are obtained by venipuncture using, for example, themarginal ear veins after dilation. The blood samples are allowed to clotovernight at 4° C. and are centrifuged at approximately 2400×g at 4° C.for about 30 minutes. The serum is removed, aliquoted and stored at 4°C. for immediate use or at -20 to -90° C. for subsequent analysis.

Serum samples from generation of polyclonal antisera or media samplesfrom production of monoclonal antisera may be analyzed for determinationof antibody titer and, in particular, for the determination of hightiter antisera Subsequently, the highest titer kringle 5 peptidefragment antisera may be tested to establish the following: a) optimalantiserum dilution for highest specific binding of the antigen andlowest non-specific binding, b) ability to bind increasing amounts ofkringle 5 peptide fragments in a standard displacement curve, c)potential cross-reactivity with related peptides and proteins includingplasminogen and kringle 5 peptide fragments of related species and d)ability to detect kringle 5 peptide fragments in cell culture media andin extracts of plasma, urine and tissues. Titer may be establishedthrough several means known in the art, such as by dot blot and densityanalysis and also by precipitation of radiolabeled peptide-antibodycomplexes using protein A, secondary antisera, cold ethanol orcharcoal-dextran followed by activity measurement with a gamma counter.If desired, the highest titer antisera may be purified on affinitycolumns. For example, kringle 5 peptide fragments may be coupled to acommercially available resin and used to form an affinity column.Antiserum samples may then be passed through the column so that kringle5 antibodies bind (via kringle 5 peptide fragments) to the column. Thesebound antibodies are subsequently eluted, collected and evaluated fordetermination of titer and specificity.

Kits for measurement of kringle 5 peptide fragments and the kringle 5receptor are also contemplated as part of the present invention.Antisera that possess the highest titer and specificity and can detectkringle 5 peptide fragments in extracts of plasma, urine, tissues andcell culture media may be used to establish assay kits for rapid,reliable, sensitive and specific measurement and localization of kringle5 peptide fragments. These assay kits may employ, but are not limitedto, the following techniques: competitive and non-competitive assays,radioimmunoassays, bioluminescence and chemilumenescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISAs, microtiter plates,immunocytochemistry and antibody-coated strips or dipsticks for rapidmonitoring of urine or blood. For each kit the range, sensitivity,precision, reliability, specificity and reproducibility of the assay areestablished by means well known to those skilled in the art.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. A kringle 5 peptide fragment RIA may beestablished in the following manner: After successful radioiodinationand purification of a kringle 5 peptide fragment, antiserum possessingthe highest titer of anti-kringle 5 peptide fragment antibodies is addedat several dilutions to tubes containing a relatively constant amount ofradioactivity, such as 10,000 cpm, in a suitable buffer system. (Bufferor preimmune serum is added to other tubes to determine non-specificbinding). After incubation at 4° C. for 24 hours, protein A is added toall tubes and the tubes are vortexed, incubated at room temperature for90 minutes and centrifuged at approximately 2000-2500×g at 4° C. toprecipitate the complexes of antibody bound to labeled antigen. Thesupernatant is removed by aspiration and radioactivity in the pelletscounted in a gamma counter. The antiserum dilution that bindsapproximately 10 to 40% of the labeled peptide after subtraction of thenon-specific binding is selected for further characterization.

Next, a dilution range (approximately 0.1 pg to 10 ng) of the kringle 5peptide fragment used for development of the antiserum is evaluated byadding known amounts of the peptide to tubes containing radiolabeledpeptide and antiserum. After an incubation period (24 or 48 hours, forexample), protein A is added and the tubes are centrifuged, thesupernatant is removed and the radioactivity in the pellet is counted.The displacement of the binding of radiolabeled the kringle 5 peptidefragment by the unlabeled kringle 5 peptide fragment (standard) providesa standard curve. Additionally, several concentrations of other kringle5 peptide fragments, plasminogens, kringle 5 peptide fragments fromdifferent species and homologous peptides may be added to the assaytubes to characterize the specificity of the kringle 5 peptide fragmentantiserum.

Thereafter, extracts of various tissues including, but not limited to,primary and secondary tumors, Lewis lung carcinoma, cultures of kringle5 peptide fragment-producing cells, placenta, uterus and other tissuessuch as brain, liver and intestine are prepared using extractiontechniques that have been successfully employed to extract kringle 5peptide fragments. After workup of the tisssue extracts, assay buffer isadded and different aliquots are placed into the RIA tubes. Extracts ofknown kringle 5 peptide fragment-producing cells produce displacementcurves that are parallel to the standard curve whereas extracts oftissues that do not produce kringle 5 peptide fragments do not displaceradiolabeled kringle 5 peptide fragments from the kringle 5 peptidefragment antiserum. Such displacement curves indicate the utility of thekringle 5 peptide fragment assay to measure kringle 5 peptide fragmentsin tissues and body fluids.

Tissue extracts that contain kringle 5 peptide fragments may also becharacterized by subjecting aliquots to reverse phase HPLC. Eluatefractions are collected, dried in Speed Vac, reconstituted in RIA bufferand analyzed in the kringle 5 RIA. In this case, the maximal amount ofkringle 5 peptide fragment immunoreactivity is located in the fractionscorresponding to the elution position of the kringle 5 peptide fragment.

The above described assay kit would provide instructions, antiserum, akringle 5 peptide fragment and possibly a radiolabeled kringle 5 peptidefragment and/or reagents for precipitation of bound kringle 5 peptidefragment/kringle 5 antibody complexes. Such a kit would be useful forthe measurement of kringle 5 peptide fragments in biological fluids andtissue extracts of animals and humans with and without tumors.

Another kit may be used to visualize or localize kringle 5 peptidefragments in tissues and cells. For example, immunohistochemistrytechniques and kits which employ such techniques are well known to thoseof ordinary skill in the art. As is known in the art, animmunohistochemistry kit would provide kringle 5 peptide fragmentantiserum; and possibly blocking serum and secondary antiserum linked toa fluorescent molecule such as fluorescein isothiocyanate or to someother reagent used to visualize the primary antiserum. Using thismethodology, biopsied tumors may be examined for sites of kringle 5peptide fragment production or for sites of the kringle 5 peptidefragment receptor. Alternatively, a kit may supply radiolabeled nucleicacids for use in in situ hybridization to probe for kringle 5 peptidefragment messenger RNA.

The compounds of the invention may be prepared using processes wellknown to those of ordinary skill in the art. (See for example,Sottrup-Jensen et al., Progress in Chemical Fibrinolysis andThrombolysis, Vol. 3, Davidson, J. F., Rowan, R. M., Samama, M. M. andDesnoyers, P. C. editors, Raven Press, New York, 1978. One manner ofpreparing kringle 5 peptide fragments is by enzymatic cleavage of thenative protein (glu-plasminogen) or a variant thereof (meaning atruncated form of the full length protein which is amenable to cleavageby enzymatic digestion and which comprises at least a kringle 5 sequenceas defined above such as lys-plasminogen or miniplasminogen). Thismethod first requires isolating the protein from human plasma in theabsence of plasmin inhibitors and thereby promoting the conversion ofglu-plasminogen to lys-plasminogen (see Novokhatny, V and Kudinov, S.A., J. Mol. Biol. 179: 215-232 (1984). Subsequently, the truncatedmolecule is treated with an proteolytic enzyme at a concentrationsufficient to cleave kringle 5 peptide fragments from the polypeptideand then purified from the remaining fragments by means known to thoseskilled in the art. A preferred proteolytic enzyme is human or porcineelastase which cleaves plasminogen and its truncated variants betweenkringle regions 3-4 and 4-5 (and is thereby capable of forming peptidefragments containing kringles 1-3 and 1-4 or kringles 4 or 5 alone). Forexample, lys-plasminogen or glu-plasminogen may be treated with porcineor human neutrophyl elastase at a ratio of about 1:100-1:300lys-plasminogen:elastase (preferably at a ratio of 1:150-1:250 and mostpreferably at a ratio of 1:150 in a buffer solution (such as Tris-HCl,NaCl, sodium phosphate and the like). Alternatively, the elastase mayfirst be immobilized (such as to a resin) to facilitate purification ofthe cleavage products. The glu-plasminogen or lys-plaminogen isgenerally treated with human or porcine elastase at temperatures rangingfrom about 10° C. to about 40° C. and for time periods ranging fromabout 4 to about 24 hours depending on the extent of cleavage desired.To achieve complete digestion of glu-plasminogen, lys-plasminogen orminiplasminogen with human or porcine elastase requires exposure ofthese polypeptides to the enzyme for at least about 12 hours at roomtemperature. Varying the pH and exposure time to the enzyme results inless or partial cleavage at one or more of the susceptible cleavagesites. The cleavage products are then purified by any means well knownin the art (such as column chromatography). A preferred purificationscheme involves applying the cleavage products to a lysine-Sepharosecolumn as described in Example 14.

Solid Phase Synthesis of Kringle 5 Peptide Fragments

The following examples will serve to further illustrate the preparationof the novel compounds of the invention:

EXAMPLE 1 N-Ac-Val-Leu-Leu-Pro-Asp-Val-Glu-Thr-Pro-Ser-Glu-Glu-Asp-NH₂

An amide peptide synthesis column (Applied Biosystems) was placed in thepeptide synthesis column position of a Perkin Elmer/Applied Biosynthesis"Synergy" peptide synthesizer, and the following synthetic sequence wasused:

1. Solvating the resin with DMF for about 5 minutes;

2. Deblocking the Fmoc group from the α-N-terminal of the resin-boundamino acid using 20% piperidine in DMF for about 15 minutes;

3. Washing the resin with DMF for about 5 minutes;

4. Activating the α-C-terminal of amino acid No. 1 (Fmoc-Asp(β-O^(t)Bu), 25 μmol) using a 0.2 M solution of HBTU (25 μmol) and HOBT (25μmol) in DMSO-NMP (N-methylpyrrolidone) and a 0.4 M solution ofdiisopropylethylamine (25 μmol) in DMSO- NMP and coupling the activatedamino acid to the resin;

5. Coupling the activated Fmoc-protected amino acid (prepared in step 5)to the resin-bound amino acid (prepared in step 2) in DMF for about 30minutes;

6. Washing with DMF for 5 minutes;

7. Repeating steps 3 through 6 with the following amino acids:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Glu(γ-O.sup.t Bu)                                  3. Fmoc-Glu(γ-O.sup.t Bu)                                               4. Fmoc-Ser(.sup.t Bu)                                                        5. Fmoc-Pro                                                                   6. Fmoc-Thr(.sup.t Bu)                                                        7. Fmoc-Glu(γ-O.sup.t Bu)                                               8. Fmoc-Val                                                                   9. Fmoc-Asp(β-O.sup.t Bu)                                                10.  Fmoc-Pro                                                                 11.  Fmoc-Leu                                                                 12.  Fmoc-Leu                                                                 13.  Fmoc-Val                                                               ______________________________________                                    

8. Coupling acetic acid to the α-N-terminal of the resin-bound peptidevia the conditions of steps 4 and 5.

9. Washing the resin with THF for about 5 minutes to remove DMF andshrink the resin, then drying the resin with argon for 10 minutes andnitrogen for 10 minutes more to provide clean, resin-bound peptide.

10. Cleaving of the peptide from the resin with concomitant deprotectionof amino acid side chains by stirring with cleavage reagent(freshly-prepared thioanisole (100 μL), water (50 μL), ethanedithiol (50μL) and trifluoroacetic acid (1.8 mL) mixed in the above order at -5° C.to -10° C.) at 0° C. for 10-15 minutes and then at ambient temperaturefor an additional 1.75 hours (plus an additional 0.5 hour for eachArg(Pmc), if present). The amount of cleavage reagent used wasdetermined by the following formula:

    ______________________________________                                        weight of resin with bound peptide (mg)                                                          amount of cleavage reagent (μL)                         ______________________________________                                         0-10              100                                                          10-25 200                                                                     25-50 400                                                                      50-100 700                                                                   100-200 1200                                                                ______________________________________                                    

11. Filtering and rinsing the produdct with neat trifluoroacetic acid,adding the filtrate in 0.5 mL portions to a centrifuge tube containingabout 8 mL of cold diethyl ether, centrifuging and decanting andrepeating the process until all of the peptide precipitated (if thepeptide did not precipitate upon addition to ether, the mixture wasextracted with aqueous 30% aqueous acetic acid (3×1 mL), and thecombined aqueous extracts were lyophilized to provide the product).

12. Using the peptide crude or purifying the peptide by HPLC using a 7μm Symmetry Prep C18 column (7.8×300 mm) with solvent mixtures varyingin a gradient from 5% to 100% acetonitrile-(water, 0.1% TFA) over aperiod of 50 minutes followed by lyophilizing to provide 35 mg ofN-Ac-Val-Leu-Leu-Pro-Asp-Val-Glu-Thr-Pro-Ser-Glu-Glu-Asp-NH₂.

EXAMPLE 2N-Ac-Met-Phe-Gly-Asn-Gly-Lys-Gly-Tyr-Arg-Gly-Lys-Arg-Ala-Thr-Thr-Val-Thr-Gly-Thr-Pro-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Pro as amino acid No. 1. The following aminoacids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                         2.               Fmoc-Thr(.sup.t Bu)                                            3. Fmoc-Gly                                                                   4. Fmoc-Thr(.sup.t Bu)                                                        5. Fmoc-Val                                                                   6. Fmoc-Thr(.sup.t Bu)                                                        7. Fmoc-Thr(.sup.t Bu)                                                        8. Fmoc-Ala                                                                   9. Fmoc-Arg(Pmc)                                                             10. Fmoc-Lys(Boc)                                                             11. Fmoc-Gly                                                                  12. Fmoc-Arg(Pmc)                                                             13. Fmoc-Tyr(.sup.t Bu)                                                       14. Fmoc-Gly                                                                  15. Fmoc-Lys(Boc)                                                             16. Fmoc-Gly                                                                  17. Fmoc-Asn(Trt)                                                             18. Fmoc-Gly                                                                  19. Fmoc-Phe                                                                  20. Fmoc-Met                                                                ______________________________________                                    

to provide 35 mg ofN-Ac-Met-Phe-Gly-Asn-Gly-Lys-Gly-Tyr-Arg-Gly-Lys-Arg-Ala-Thr-Thr-Val-Thr-Gly-Thr-Pro-NH₂.

EXAMPLE 3Ac-Gln-Asp-Trp-Ala-Ala-Gln-Glu-Pro-His-Arg-His-Ser-Ile-Phe-Thr-Pro-Glu-Thr-Asn-Pro-Arg-Ala-Gly-Leu-Glu-Lys-Asn-Tyr-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                         2.               Fmoc-Asn(Trt)                                                  3. Fmoc-Lys(Boc)                                                              4. Fmoc-Glu(γ-O.sup.t Bu)                                               5. Fmoc-Leu                                                                   6. Fmoc-Gly                                                                   7. Fmoc-Ala                                                                   8. Fmoc-Arg(Pmc)                                                              9. Fmoc-Pro                                                                  10. Fmoc-Asn(Trt)                                                             11. Fmoc-Thr(.sup.t Bu)                                                       12. Fmoc-Glu(γ-O.sup.t Bu)                                              13. Fmoc-Pro                                                                  14. Fmoc-Thr(.sup.t Bu)                                                       15. Fmoc-Phe                                                                  16. Fmoc-Ile                                                                  17. Fmoc-Ser(.sup.t Bu)                                                       18. Fmoc-His(Trt)                                                             19. Fmoc-Arg(Pmc)                                                             20. Fmoc-His(Trt)                                                             21. Fmoc-Pro                                                                  22. Fmoc-Glu(γ-O.sup.t Bu)                                              23. Fmoc-Gln(Trt)                                                             24. Fmoc-Ala                                                                  25. Fmoc-Ala                                                                  26. Fmoc-Trp                                                                  27. Fmoc-Asp(β-O.sup.t Bu)                                               28. Fmoc-Gln(Trt)                                                           ______________________________________                                    

to provide 40 mg ofN-Ac-Gln-Asp-Trp-Ala-Ala-Gln-Glu-Pro-His-Arg-His-Ser-Ile-Phe-Thr-Pro-Glu-Thr-Asn-Pro-Arg-Ala-Gly-Leu-Glu-Lys-Asn-Tyr-NH₂.

EXAMPLE 4 N-Ac-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Trp as amino acid No. 1. The following aminoacids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Pro                                                      3. Fmoc-Gly                                                                   4. Fmoc-Gly                                                                   5. Fmoc-Val                                                                   6. Fmoc-Asp(β-O.sup.t -Bu)                                               7. Fmoc-Gly                                                                   8. Fmoc-Asp(β-O.sup.t -Bu)                                               9. Fmoc-Pro                                                                   10.  Fmoc-Asn(Trt)                                                            11.  Fmoc-Arg(Pmt)                                                          ______________________________________                                    

to provide 20 mg ofN-Ac-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-NH₂.

EXAMPLE 5 N-Ac-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂

The title compound was prepared using the synthetic sequence describedin example 1 and using Fmoc-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Asp(β- O.sup.t Bu)                                  3. Fmoc-Tyr(.sup.t Bu)                                                        4. Fmoc-Leu                                                                   5. Fmoc-Lys(Boc)                                                              6. Fmoc-Arg(Pmc)                                                              7. Fmoc-Pro                                                                   8. Fmoc-Asn(Trt)                                                              9. Fmoc-Thr(.sup.t Bu)                                                        10.  Fmoc-Thr(.sup.t Bu)                                                      11.  Fmoc-Tyr(.sup.t Bu)                                                    ______________________________________                                    

to provide 10 mg ofN-Ac-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.

EXAMPLE 6 N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Asp(β-O.sup.t Bu)                                   3. Fmoc-Tyr(.sup.t Bu)                                                        4. Fmoc-Leu                                                                   5. Fmoc-Lys(Boc)                                                              6. Fmoc-Arg(Pmc)                                                              7. Fmoc-Pro                                                                 ______________________________________                                    

to provide N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂ (4 mg). MS (FAB) m/z 995(M+H)⁺.

EXAMPLE 7 N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1. The following amino acids were added using the conditionsindicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Tyr(.sup.t Bu)                                           3. Fmoc-Leu                                                                   4. Fmoc-Lys(Boc)                                                              5. Fmoc-Arg(Pmc)                                                              6. Fmoc-Leu                                                                 ______________________________________                                    

to provide N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-NH₂ (6 mg). MS (ESI) m/z 832(M+H)⁺.

EXAMPLE 8 N-Ac-Pro-Glu-Lys-Arg-Tyr-Asp-Tyr-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Asp(β-O.sup.t Bu)                                   3. Fmoc-Tyr(.sup.t Bu)                                                        4. Fmoc-Arg(Pmc)                                                              5. Fmoc-Lys(Boc)                                                              6. Fmoc-Glu                                                                   7. Fmoc-Pro                                                                 ______________________________________                                    

to provide N-Ac-Pro-Glu-Lys-Arg-Tyr-Asp-Tyr-NH₂ (6 mg). MS (FAB) m/z(1101) (M+H)⁺.

EXAMPLE 9 N-Ac-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Asp(β-O.sup.t Bu)                                   3. Fmoc-Tyr(.sup.t Bu)                                                        4. Fmoc-Leu                                                                   5. Fmoc-Lys(Boc)                                                              6. Fmoc-Arg(Pmc)                                                            ______________________________________                                    

to provide N-Ac-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂ (8 mg). MS (ESI) m/z 898(M+H)⁺.

EXAMPLE 10 N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH₂ (SEQ ID NO: 13)

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Asp(β-O.sup.t Bu)                                   3. Fmoc-3-I-Tyr(.sup.t Bu)                                                    4. Fmoc-Leu                                                                   5. Fmoc-Lys(Boc)                                                              6. Fmoc-Arg(Pmc)                                                              7. Fmoc-Pro                                                                 ______________________________________                                    

to provide N-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH₂ (2 mg). MS (ESI) m/z(1121) (M+H)⁺.

EXAMPLE 11 N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH₂ (SEQ ID NO: 14)

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-3-I-Tyr(^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.               Amino Acid                                                  ______________________________________                                        2.                Fmoc-Asp(β-O.sup.t Bu)                                   3. Fmoc-Tyr(.sup.t Bu)                                                        4. Fmoc-Leu                                                                   5. Fmoc-Lys(Boc)                                                              6. Fmoc-Arg(Pmc)                                                              7. Fmoc-Pro                                                                 ______________________________________                                    

to provide N-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH₂ (2.5 mg). MS (ESI)m/z 1121 (M+H)⁺.

EXAMPLE 12 N-Ac-Lys-Leu-Tyr-Asp-NH₂

The title compound was prepared using the synthetic sequence describedin Example 1 and using Fmoc-Asp(β-O^(t) Bu) as amino acid No. 1. Thefollowing amino acids were added using the conditions indicated:

    ______________________________________                                        No.                Amino Acid                                                 ______________________________________                                        2.                 Fmoc-Tyr(.sup.t Bu)                                          3. Fmoc-Leu                                                                   4. Fmoc-Lys                                                                 ______________________________________                                    

to provide 2 mg of N-Ac-Lys-Leu-Tyr-Asp-NH₂ (2 mg).

EXAMPLE 13 Preparation and separation of a mixtureN-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I¹²⁵ -Tyr⁵³⁵ -NH₂ andN-Ac-Pro-Arg-Lys-Leu-3-I¹²⁵ -Tyr⁵³³ -Asp-Tyr-NH₂ (SEQ ID NO: 13) and(SEQ NO: ID 14), Respectively

To a solution of 30 μg ofN-acetyl-prolyl-arginyl-lysyl-leucyl-tyrosyl-aspartyl-tyrosylamide in 80mL of phosphate buffered saline (PBS) was added one iodobead (Pierce,Rockford, Ill.) and 100 μCi of NaI¹²⁵. After 10 minutes, the excessNaI¹²⁵ reagent was removed by applying the reaction mixture to a WatersC18-Light SepPack column and eluting with water then 0.1% TFA in 1:1 CH₃CN/water and collecting 3×200 μL fractions to provide a mixture ofTyr⁵³³ - and Tyr⁵³⁵ - radiolabeled peptides.

The hot peptide mixture was coinjected onto a C18 HPLC column with anequimolar solution of cold carriersN-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH₂ andN-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH₂, the elution times of which hadbeen predetermined as 36 and 38 minutes, respectively. Repeated elutionswith the solvent system in Example 1 and lyophylization of the combined,relevant fractions provided the desired compoundN-Ac-Pro-Arg-Lys-Leu-Tyr-Asp-3-I-Tyr-NH₂ with a minimal impurityN-Ac-Pro-Arg-Lys-Leu-3-I-Tyr-Asp-Tyr-NH₂.

General Methodologies EXAMPLE 14 Isolation and Purification of Kringle 5Peptide Fragments

The kringle 5 peptide fragments were prepared from the digestion of Lysplasminogen (Lys-HPg, Abbott Laboratories, Abbott Park, Ill.) withporcine elastase (SIGMA, St. Louis, Mo.) by a modification of the methodof Powell et al. (Arch Biochem. Biophys. 248 (1): 390-400 (1986), whichis hereby incorporated herein by reference). 1.5 mg of porcine elastasewas incubated with 200 mg of Lys-HPg in 50 mM Tris-HCl pH 8.0 and rockedovernight at room temperature. The reaction was terminated by theaddition of DPF (diisopropyl fluorophosphate, SIGMA) to a finalconcentration of 1 mM. The mixture was rocked for an additional 30minutes, dialysed against 50 mM Tris pH 8.0 overnight and concentrated.The cleaved plasminogen was placed over a 2.5 cm×15 cm lysine-Sepharose4B column (Brockway, W. J. and Castellino, F. J., Arch. Biochem.Biophys. 151: 194-199 (1972), which is hereby incorporated by reference)and equilibrated with 50 mM Tris pH 8.0 until an absorbance of 0.05 (at280 nm) was reached. (This step was performed to remove any fragmentscontaining a kringle 1 region and/or a kringle 4 region (both of whichbind lysine)). The non-absorbed kringle 5 peptide fragments weredialysed against 50 mM Na₂ PO₄ buffer, pH 5.0 then applied to a BioRadMono-S column equilibrated with the same buffer. The cleaved kringle 5portion, uncut mini-HPg and remaining protease domain fraction wereeluted with a 0-20%, 20-50% and 50-70% step gradient of 20 mMPhosphate/1 M KCl pH 5.0. The kringle 5 peptide fragments eluted at the50% step as determined by gel electrophesis. The collected peak wasdialysed overnight against 20 mM Tris pH 8.0.

The separated kringle 5 fragments were determined to be at least 95%pure by FPLC chromatography and DodSO4/PAGE with silver staining(Coomasie Blue). Sequence analysis of the amino terminal portion of thepurified fragments revealed the presence of three polypeptides havingα-N-terminus sequences of VLLPDVETPS, VAPPPVVLL and VETPSEED whchcorrespond to amino acid positions Val⁴⁴⁹ -Ser⁴⁵⁸, Val⁴⁴³ -Leu⁴⁵⁰ andVal⁴⁵⁴ -Asp⁴⁶¹ of SEQ ID. NO: 1, respectively.

EXAMPLE 15 Endothelial Proliferation Assay

The in vitro proliferation of endothelial cells was determined asdescribed by Lingen, et al., in Laboratory Investigation, 74: 476-483(1996), which is hereby incorporated herein by reference, using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit (PromegaCorporation, Madison, Wis.). Bovine capillary (adrenal) endothelialcells were plated at a density of 1000 cells per well in a 96-well platein Dulbecco's Modified Eagle Medium (DMEM) containing 10% donor calfserum and 1% BSA (bovine serum albumin, GIBCO BRL, Gaithersburg, Md.).After 8 hours, the cells were starved overnight in DMEM containing 0.1%BSA then re-fed with media containing specified concentrations ofinhibitor and 5 ng/mL bFGF (basic fibroblast growth factor). The resultsof the assay were corrected both for unstimulated cells (i.e. no bFGFadded) as the baseline and for cells stimulated with bFGF alone (i.e. noinhibitor added) as the maximal proliferation. When multple experimentswere combined, the results were represented as the percent change incell number as compared to bFGF alone.

EXAMPLE 16 Endothelial Cell Migration Assay

The endothelial cell migration assay was performed essentially asdescribed by Polverini, P. J. et al., Methods Enzymol, 198: 440-450(1991), which is hereby incorporated herein by reference. Briefly,bovine capillary (adrenal) endothelial cells (BCE, supplied by JudahFolkman, Harvard University Medical School) were starved overnight inDMEM containing 0.1% bovine serum albumin (BSA). Cells were thenharvested with trypsin and resuspended in DMEM with 0.1% BSA at aconcentration of 1.5×10⁶ cells/mL. Cells were added to the bottom of a48-well modified Boyden chamber (Nucleopore Corporation, Cabin John,Md.). The chamber was assembled and inverted, and cells were allowed toattach for 2 hours at 37° C. to polycarbonate chemotaxis membranes (5 μmpore size) that had been soaked in 0.1% gelatin overnight and dried. Thechamber was then reinverted and test substances were added to the wellsof the upper chamber (to a total volume of 50 μL); the apparatus wasthen incubated for 4 hours at 37° C. Membranes were recovered, fixed andstained (DiffQuick, Fisher Scientific, Pittsburgh, Pa.) and the numberof cells that had migrated to the upper chamber per 10 high power fieldswere counted. Background migration to DMEM+0.1% BSA was subtracted andthe data reported as the number of cells migrated per 10 high powerfields (400X) or when results from multiple experiments were combined,as the percent inhibition of migration compared to a positive control.The results are shown in Table 1.

EXAMPLE 17 Effect of Kringle 5 Peptide Fragments on Endothetial CellProliferation in vitro

The effect of kringle 5 peptide fragments on endothelial cellproliferation was determined in vitro using the above describedendothelial cell proliferation assay. For these experiments, kringle 5peptide fragments was prepared as illustrated in Examples 1 through 14and tested at various concentrations ranging from about 100 to 1000 pMwith bFGF used as a maximum proliferation control. The kringle 5 peptidefragment SEQ ID NO: 3 was effective at inhibiting BCE cell proliferationin a dose-dependent manner. The concentration of kringle 5 peptidefragment SEQ ID NO: 3 required to reach 50% inhibition (ED₅₀) wasdetermined at about 300 pM. In contrast, the ED₅₀ of kringles 1-4 wasshown to be 135 nM.

A summary of the effect of other kringle peptide fragments on inhibitionof BCE cell proliferation is shown in Table 1. The kringle 3 peptidefragment was least effective at inhibitng BCE cell proliferation (ED₅₀=460 nM), followed by the kringle 1 peptide fragment (ED₅₀ =320 nM),kringle 1-4 peptide fragments (ED₅₀ =135 nM) and kringles 1-3 peptidefragments (ED₅₀ =75 nM). The kringle 5 peptide fragment was the mosteffective at inhibiting BCE cell proliferation with an ED₅₀ of 0.3 nM.

EXAMPLE 18 Effect of Kringle 5 Peptide Fragments on Endothelial CellMigration in vito

The effect of kringle 5 peptide fragments on endothelial cell migrationwas also determined in vitro using the above described endothelial cellmigration assay. Kringle 5 peptide fragments inhibited BCE cellmigration in a dose-dependent fashion with an ED₅₀ of approximately 300pM. At the concentration of kringle 5 peptide fragments required formaximal inhibition of BCE cells, PC-3 cells and MDA 486 cells were alsoinhibited. This result, taken together with the result in Example 2,indicates that the inhibition of stimulated proliferation and migrationof BCE cells by kringle 5 peptide fragments is both potent and specificto endothelial cells and not to normal or tumor cells.

The foregoing are merely illustrative of the invention and are notintended to limit the invention to the disclosed compounds. Variationsand changes which are obvious to one skilled in the art are intended tobe within the scope and nature of the invention which are defined in theappended claims.

Table 1 shows a summary of ED₅₀ values obtained from the inhibition ofvarious kringle fragments on BCE cell proliferation and cell migrationin vitro. In the table, kringle peptide fragments are labeled accordingto their corresponding sequence homology to SEQ ID NO: 1. The symbol "*"indicates data taken from Marti, D., et al., Eur. J. Biochem., 219:455-462 (1994), which is hereby incorporated by reference.

                  TABLE 1                                                         ______________________________________                                                          Antiproliferative                                                                         Migratory                                          Activity of Inhibition of                                                    Protein Fragment from BCE Cells HMVEC Cells                                   SEQ ID NO:1 (ED.sub.50) (ED.sub.50)                                         ______________________________________                                        kringles 1-4(angiostatin)*                                                                      135 nM      160    nM                                         kringle 1 (Tyr.sup.80 -Glu.sup.163)* 320 nM --                                kringle 2 (Glu.sup.161 -Thr.sup.245)* no activity --                          kringle 3 (Thr.sup.253 -Ser.sup.335)* 460 nM --                               kringle 4 (Val.sup.354 -Val.sup.443)* no activity --                          kringles 1-3 (Tyr.sup.80 -Pro.sup.353)*  75 nM 60 nM                          kringles 2-3 (Glu.sup.161 -Ser.sup.335)* -- --                                kringle 5 (Val.sup.443 -Ala.sup.543) 250 pM 200 pM                            kringle 5 (Val.sup.449 -Ala.sup.543) -- 240 pM                                kringle 5 (Val.sup.454 -Ala.sup.543) -- 220 pM                                kringle 5 (Val.sup.443 -Phe.sup.546)  60 nM 55 nM                             kringle 5 (Val.sup.449 -Phe.sup.546) -- --                                    kringle 5 (Val.sup.454 -Phe.sup.546) -- --                                    kringles 4-5 (Val.sup.355 -Ala.sup.543) -- 280 pM                             kringles 4-5 (Val.sup.355 -Phe.sup.546) -- --                                 N-Ac-Val.sup.449 -Asp.sup.461 -NH.sub.2 -- >1 mM                              N-Ac-Met.sup.463 -Pro.sup.482 -NH.sub.2 -- >1 mM                              N-Ac-Gln.sup.484 -Tyr.sup.511 -NH.sub.2 -- >100 μM                         N-Ac-Arg.sup.513 -Trp.sup.523 -NH.sub.2 -- 500 pM                             N-Ac-Tyr.sup.525 -Trp.sup.535 -NH.sub.2 -- 200 pM                             N-Ac-Pro.sup.529 -Tyr.sup.535 -NH.sub.2 -- 120 pM                             N-Ac-Pro.sup.529 -Asp.sup.534 -NH.sub.2 -- 123 pM                             N-Ac-Pro.sup.150 -Tyr.sup.156 -NH.sub.2 -- 160 nM                             N-Ac-Arg.sup.530 -Tyr.sup.535 -NH.sub.2 -- 80 nM                              N-Ac-Pro-Arg-Lys-Leu-3-I- -- >100 nM                                          Tyr-Asp-Tyr-NH.sub.2                                                          N-Ac-Pro-Arg-Lys-Leu-Tyr- -- 400 pM                                           Asp-3-I-Tyr-NH.sub.2                                                          N-Ac-Lys.sup.531 -Tyr.sup.534 -NH.sub.2 -- --                               ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 14                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 791 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:1:                    - - Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gl - #y Ala Ser Leu Phe        Ser                                                                              1               5  - #                 10 - #                 15             - - Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Il - #e Glu Glu Cys Ala Ala                   20     - #             25     - #             30                  - - Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Ar - #g Ala Phe Gln Tyr His               35         - #         40         - #         45                      - - Ser Lys Glu Gln Gln Cys Val Ile Met Ala Gl - #u Asn Arg Lys Ser Ser           50             - #     55             - #     60                          - - Ile Ile Ile Arg Met Arg Asp Val Val Leu Ph - #e Glu Lys Lys Val Tyr       65                 - # 70                 - # 75                 - # 80       - - Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys As - #n Tyr Arg Gly Thr Met                       85 - #                 90 - #                 95              - - Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Ly - #s Trp Ser Ser Thr Ser                  100      - #           105      - #           110                  - - Pro His Arg Pro Arg Phe Ser Pro Ala Thr Hi - #s Pro Ser Glu Gly Leu              115          - #       120          - #       125                      - - Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn As - #p Pro Gln Gly Pro Trp          130              - #   135              - #   140                          - - Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr As - #p Tyr Cys Asp Ile Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Cys Glu Glu Glu Cys Met His Cys Ser Gl - #y Glu Asn Tyr Asp        Gly                                                                                             165  - #               170  - #               175             - - Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cy - #s Gln Ala Trp Asp Ser                  180      - #           185      - #           190                  - - Gln Ser Pro His Ala His Gly Tyr Ile Pro Se - #r Lys Phe Pro Asn Lys              195          - #       200          - #       205                      - - Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro As - #p Arg Glu Leu Arg Pro          210              - #   215              - #   220                          - - Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Tr - #p Glu Leu Cys Asp Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gl - #y Pro Thr Tyr Gln        Cys                                                                                             245  - #               250  - #               255             - - Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly As - #n Val Ala Val Thr Val                  260      - #           265      - #           270                  - - Ser Gly His Thr Cys Gln His Trp Ser Ala Gl - #n Thr Pro His Thr His              275          - #       280          - #       285                      - - Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys As - #n Leu Asp Glu Asn Tyr          290              - #   295              - #   300                          - - Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Tr - #p Cys His Thr Thr Asn      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pr - #o Ser Cys Asp Ser        Ser                                                                                             325  - #               330  - #               335             - - Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Al - #a Pro Pro Glu Leu Thr                  340      - #           345      - #           350                  - - Pro Val Val Gln Asp Cys Tyr His Gly Asp Gl - #y Gln Ser Tyr Arg Gly              355          - #       360          - #       365                      - - Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cy - #s Gln Ser Trp Ser Ser          370              - #   375              - #   380                          - - Met Thr Pro His Arg His Gln Lys Thr Pro Gl - #u Asn Tyr Pro Asn Ala      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro As - #p Ala Asp Lys Gly        Pro                                                                                             405  - #               410  - #               415             - - Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Tr - #p Glu Tyr Cys Asn Leu                  420      - #           425      - #           430                  - - Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Va - #l Ala Pro Pro Pro Val              435          - #       440          - #       445                      - - Val Leu Leu Pro Asp Val Glu Thr Pro Ser Gl - #u Glu Asp Cys Met Phe          450              - #   455              - #   460                          - - Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Al - #a Thr Thr Val Thr Gly      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pr - #o His Arg His Ser        Ile                                                                                             485  - #               490  - #               495             - - Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Le - #u Glu Lys Asn Tyr Cys                  500      - #           505      - #           510                  - - Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Tr - #p Cys Tyr Thr Thr Asn              515          - #       520          - #       525                      - - Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pr - #o Gln Cys Ala Ala Pro          530              - #   535              - #   540                          - - Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pr - #o Lys Lys Cys Pro Gly      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Arg Val Val Gly Gly Cys Val Ala His Pro Hi - #s Ser Trp Pro Trp        Gln                                                                                             565  - #               570  - #               575             - - Val Ser Leu Arg Thr Arg Phe Gly Met His Ph - #e Cys Gly Gly Thr Leu                  580      - #           585      - #           590                  - - Ile Ser Pro Glu Trp Val Leu Thr Ala Ala Hi - #s Cys Leu Glu Lys Ser              595          - #       600          - #       605                      - - Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gl - #y Ala His Gln Glu Val          610              - #   615              - #   620                          - - Asn Leu Glu Pro His Val Gln Glu Ile Glu Va - #l Ser Arg Leu Phe Leu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Ly - #s Leu Ser Ser Pro        Ala                                                                                             645  - #               650  - #               655             - - Val Ile Thr Asp Lys Val Ile Pro Ala Cys Le - #u Pro Ser Pro Asn Tyr                  660      - #           665      - #           670                  - - Val Val Ala Asp Arg Thr Glu Cys Phe Ile Th - #r Gly Trp Gly Glu Thr              675          - #       680          - #       685                      - - Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Gl - #u Ala Gln Leu Pro Val          690              - #   695              - #   700                          - - Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Ph - #e Leu Asn Gly Arg Val      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Gln Ser Thr Glu Leu Cys Ala Gly His Leu Al - #a Gly Gly Thr Asp        Ser                                                                                             725  - #               730  - #               735             - - Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cy - #s Phe Glu Lys Asp Lys                  740      - #           745      - #           750                  - - Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Le - #u Gly Cys Ala Arg Pro              755          - #       760          - #       765                      - - Asn Lys Pro Gly Val Tyr Val Arg Val Ser Ar - #g Phe Val Thr Trp Ile          770              - #   775              - #   780                          - - Glu Gly Val Met Arg Asn Asn                                              785                 7 - #90                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:2:                    - - Val Ala Pro Pro Pro Val Val Leu Leu Pro As - #p Val Glu Thr Pro Ser      1               5   - #               10   - #               15                - - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gl - #y Tyr Arg Gly Lys Arg                  20      - #            25      - #            30                   - - Ala Thr Thr Val Thr Gly Thr Pro Cys Gln As - #p Trp Ala Ala Gln Glu              35          - #        40          - #        45                       - - Pro His Arg His Ser Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly          50              - #    55              - #    60                           - - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Gly Gly Pro      65                  - #70                  - #75                  - #80        - - Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Ty - #r Asp Tyr Cys Asp Val                      85  - #                90  - #                95               - - Pro Gln Cys Ala Ala                                                                  100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION: protein                                             - -      (v) FRAGMENT TYPE:  internal                                         - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:3:                    - - Val Leu Leu Pro Asp Val Glu Thr Pro Ser Gl - #u Glu Asp Cys Met Phe       1               5  - #                10  - #                15               - - Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Al - #a Thr Thr Val Thr Gly                  20      - #            25      - #            30                   - - Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pr - #o His Arg His Ser Ile              35          - #        40          - #        45                       - - Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Le - #u Glu Lys Asn Tyr Cys          50              - #    55              - #    60                           - - Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Tr - #p Cys Tyr Thr Thr Asn      65                  - #70                  - #75                  - #80        - - Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pr - #o Gln Cys Ala Ala                          85  - #                90  - #                95               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:4:                    - - Val Glu Thr Pro Ser Glu Glu Asp Cys Met Ph - #e Gly Asn Gly Lys Gly      1               5   - #               10   - #               15                - - Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gl - #y Thr Pro Cys Gln Asp                  20      - #            25      - #            30                   - - Trp Ala Ala Gln Glu Pro His Arg His Ser Il - #e Phe Thr Pro Glu Thr              35          - #        40          - #        45                       - - Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cy - #s Arg Asn Pro Asp Gly          50              - #    55              - #    60                           - - Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr As - #n Pro Arg Lys Leu Tyr      65                  - #70                  - #75                  - #80        - - Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala                                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:5:                    - - Val Ala Pro Pro Pro Val Val Leu Leu Pro As - #p Val Glu Thr Pro Ser      1               5   - #               10   - #               15                - - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gl - #y Tyr Arg Gly Lys Arg                  20      - #            25      - #            30                   - - Ala Thr Thr Val Thr Gly Thr Pro Cys Gln As - #p Trp Ala Ala Gln Glu              35          - #        40          - #        45                       - - Pro His Arg His Ser Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly          50              - #    55              - #    60                           - - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Gly Gly Pro      65                  - #70                  - #75                  - #80        - - Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Ty - #r Asp Tyr Cys Asp Val                      85  - #                90  - #                95               - - Pro Gln Cys Ala Ala Pro Ser Phe                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:6:                    - - Val Leu Leu Pro Asp Val Glu Thr Pro Ser Gl - #u Glu Asp Cys Met Phe      1               5   - #               10   - #               15                - - Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Al - #a Thr Thr Val Thr Gly                  20      - #            25      - #            30                   - - Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pr - #o His Arg His Ser Ile              35          - #        40          - #        45                       - - Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Le - #u Glu Lys Asn Tyr Cys          50              - #    55              - #    60                           - - Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Tr - #p Cys Tyr Thr Thr Asn      65                  - #70                  - #75                  - #80        - - Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pr - #o Gln Cys Ala Ala Pro                      85  - #                90  - #                95               - - Ser Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:7:                    - - Val Glu Thr Pro Ser Glu Glu Asp Cys Met Ph - #e Gly Asn Gly Lys Gly      1               5   - #               10   - #               15                - - Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gl - #y Thr Pro Cys Gln Asp                  20      - #            25      - #            30                   - - Trp Ala Ala Gln Glu Pro His Arg His Ser Il - #e Phe Thr Pro Glu Thr               35         - #         40         - #         45                      - - Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cy - #s Arg Asn Pro Asp Gly          50              - #    55              - #    60                           - - Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr As - #n Pro Arg Lys Leu Tyr      65                  - #70                  - #75                  - #80        - - Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pr - #o Ser Phe                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY:  mouse - #sequence                                     - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:8:                    - - Val Glu Leu Pro Thr Val Ser Gln Glu Pro Se - #r Gly Pro Ser Asp Ser      1               5   - #               10   - #               15                - - Glu Thr Asp Cys Met Tyr Gly Asn Asp Lys As - #p Tyr Arg Thr Lys Thr                  20      - #            25      - #            30                   - - Ala Val Ala Ala Ala Gly Thr Pro Gly Gln Gl - #y Trp Ala Ala Gln Glu              35          - #        40          - #        45                       - - Pro His Arg His Ser Ile Phe Thr Pro Gln Th - #r Asn Pro Arg Ala Gly          50              - #    55              - #    60                           - - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Asn Gly Pro      65                  - #70                  - #75                  - #80        - - Trp Cys Tyr Thr Thr Asn Pro Arg Ser Leu Ty - #r Asp Tyr Cys Asp Ile                        - # 85                 - # 90                 - # 95         - - Pro Leu Cys Ala Ser Ala                                                              100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY:  monkey - #sequence                                    - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:9:                    - - Ala Ala Pro Pro Pro Val Ala Gln Leu Pro As - #p Ala Glu Thr Pro Ser      1               5   - #               10   - #               15                - - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gl - #y Tyr Arg Gly Lys Lys                  20      - #            25      - #            30                   - - Ala Thr Thr Val Thr Gly Thr Pro Cys Gln As - #p Trp Ala Ala Gln Glu              35          - #        40          - #        45                       - - Pro His Ser His Arg Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly          50              - #    55              - #    60                           - - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Val Gly Gly Pro      65                  - #70                  - #75                  - #80        - - Trp Cys Tyr Thr Thr Asn Pro Arg Ser Leu Ph - #e Asp Tyr Cys Asp Val                      85  - #                90  - #                95               - - Pro Gln Cys Ala                                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: bovine se - #quence                                    - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:10:                   - - Pro Ala Ala Pro Gln Ala Pro Gly Val Glu As - #n Pro Pro Glu Ala Asp      1               5   - #               10   - #               15                - - Cys Met Ile Gly Thr Gly Lys Ser Tyr Arg Gl - #y Lys Lys Ala Thr Thr                  20      - #            25      - #            30                   - - Val Ala Gly Val Pro Cys Gln Glu Trp Ala Al - #a Gln Glu Pro His His              35          - #        40          - #        45                       - - His Ser Ile Phe Thr Pro Glu Thr Asn Pro Gl - #n Ser Gly Leu Glu Arg          50              - #    55              - #    60                           - - Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val As - #n Gly Pro Trp Cys Tyr      65                  - #70                  - #75                  - #80        - - Thr Met Asn Pro Arg Ser Leu Phe Asp Tyr Cy - #s Asp Val Pro Gln Cys                      85  - #                90  - #                95               - - Glu                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  prote - #in                                        - -      (v) FRAGMENT TYPE:  internal                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: porcine s - #equence                                   - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:11:                   - - Thr Asn Phe Pro Ala Ile Ala Gln Val Pro Se - #r Val Glu Asp Leu Ser      1               5   - #               10   - #               15                - - Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Ar - #g Tyr Arg Gly Lys Arg                   20     - #             25     - #             30                  - - Ala Thr Thr Val Ala Gly Val Pro Cys Gln Gl - #u Trp Ala Ala Gln Glu              35          - #        40          - #        45                       - - Pro His Arg His Ser Ile Phe Thr Pro Glu Th - #r Asn Pro Arg Ala Gly          50              - #    55              - #    60                           - - Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gl - #y Asp Asp Asn Gly Pro      65                  - #70                  - #75                  - #80        - - Trp Cys Tyr Thr Thr Asn Pro Gln Lys Leu Ph - #e Asp Tyr Cys Asp Val                      85  - #                90  - #                95               - - Pro Gln Cys Val Thr                                                                  100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2497 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:12:                   - - CATCCTGGGA TTGGGACCCA CTTTCTGGGC ACTGCTGGCC AGTCCCAAAA TG -             #GAACATAA     60                                                                 - - GGAAGTGGTT CTTCTACTTC TTTTATTTCT GAAATCAGGT CAAGGAGAGC CT -            #CTGGATGA    120                                                                 - - CTATGTGAAT ACCCAGGGGG CTTCACTGTT CAGTGTCACT AAGAAGCAGC TG -            #GGAGCAGG    180                                                                 - - AAGTATAGAA GAATGTGCAG CAAAATGTGA GGAGGACGAA GAATTCACCT GC -            #AGGGCATT    240                                                                 - - CCAATATCAC AGTAAAGAGC AACAATGTGT GATAATGGCT GAAAACAGGA AG -            #TCCTCCAT    300                                                                 - - AATCATTAGG ATGAGAGATG TAGTTTTATT TGAAAAGAAA GTGTATCTCT CA -            #GAGTGCAA    360                                                                 - - GACTGGGAAT GGAAAGAACT ACAGAGGGAC GATGTCCAAA ACAAAAAATG GC -            #ATCACCTG    420                                                                 - - TCAAAAATGG AGTTCCACTT CTCCCCACAG ACCTAGATTC TCACCTGCTA CA -            #CACCCCTC    480                                                                 - - AGAGGGACTG GAGGAGAACT ACTGCAGGAA TCCAGACAAC GATCCGCAGG GG -            #CCCTGGTG    540                                                                 - - CTATACTACT GATCCAGAAA AGAGATATGA CTACTGCGAC ATTCTTGAGT GT -            #GAAGAGGA    600                                                                 - - ATGTATGCAT TGCAGTGGAG AAAACTATGA CGGCAAAATT TCCAAGACCA TG -            #TCTGGACT    660                                                                 - - GGAATGCCAG GCCTGGGACT CTCAGAGCCC ACACGCTCAT GGATACATTC CT -            #TCCAAATT    720                                                                 - - TCCAAACAAG AACCTGAAGA AGAATTACTG TCGTAACCCC GATAGGGAGC TG -            #CGGCCTTG    780                                                                 - - GTGTTTCACC ACCGACCCCA ACAAGCGCTG GGAACTTTGT GACATCCCCC GC -            #TGCACAAC    840                                                                 - - ACCTCCACCA TCTTCTGGTC CCACCTACCA GTGTCTGAAG GGAACAGGTG AA -            #AACTATCG    900                                                                 - - CGGGAATGTG GCTGTTACCG TGTCCGGGCA CACCTGTCAG CACTGGAGTG CA -            #CAGACCCC    960                                                                 - - TCACACACAT AACAGGACAC CAGAAAACTT CCCCTGCAAA AATTTGGATG AA -            #AACTACTG   1020                                                                 - - CCGCAATCCT GACGGAAAAA GGGCCCCATG GTGCCATACA ACCAACAGCC AA -            #GTGCGGTG   1080                                                                 - - GGAGTACTGT AAGATACCGT CCTGTGACTC CTCCCCAGTA TCCACGGAAC AA -            #TTGGCTCC   1140                                                                 - - CACAGCACCA CCTGAGCTAA CCCCTGTGGT CCAGGACTGC TACCATGGTG AT -            #GGACAGAG   1200                                                                 - - CTACCGAGGC ACATCCTCCA CCACCACCAC AGGAAAGAAG TGTCAGTCTT GG -            #TCATCTAT   1260                                                                 - - GACACCACAC CGGCACCAGA AGACCCCAGA AAACTACCCA AATGCTGGCC TG -            #ACAATGAA   1320                                                                 - - CTACTGCAGG AATCCAGATG CCGATAAAGG CCCCTGGTGT TTTACCACAG AC -            #CCCAGCGT   1380                                                                 - - CAGGTGGGAG TACTGCAACC TGAAAAAATG CTCAGGAACA GAAGCGAGTG TT -            #GTAGCACC   1440                                                                 - - TCCGCCTGTT GTCCTGCTTC CAGATGTAGA GACTCCTTCC GAAGAAGACT GT -            #ATGTTTGG   1500                                                                 - - GAATGGGAAA GGATACCGAG GCAAGAGGGC GACCACTGTT ACTGGGACGC CA -            #TGCCAGGA   1560                                                                 - - CTGGGCTGCC CAGGAGCCCC ATAGACACAG CATTTTCACT CCAGAGACAA AT -            #CCACGGGC   1620                                                                 - - GGGTCTGGAA AAAAATTACT GCCGTAACCC TGATGGTGAT GTAGGTGGTC CC -            #TGGTGCTA   1680                                                                 - - CACGACAAAT CCAAGAAAAC TTTACGACTA CTGTGATGTC CCTCAGTGTG CG -            #GCCCCTTC   1740                                                                 - - ATTTGATTGT GGGAAGCCTC AAGTGGAGCC GAAGAAATGT CCTGGAAGGG TT -            #GTAGGGGG   1800                                                                 - - GTGTGTGGCC CACCCACATT CCTGGCCCTG GCAAGTCAGT CTTAGAACAA GG -            #TTTGGAAT   1860                                                                 - - GCACTTCTGT GGAGGCACCT TGATATCCCC AGAGTGGGTG TTGACTGCTG CC -            #CACTGCTT   1920                                                                 - - GGAGAAGTCC CCAAGGCCTT CATCCTACAA GGTCATCCTG GGTGCACACC AA -            #GAAGTGAA   1980                                                                 - - TCTCGAACCG CATGTTCAGG AAATAGAAGT GTCTAGGCTG TTCTTGGAGC CC -            #ACACGAAA   2040                                                                 - - AGATATTGCC TTGCTAAAGC TAAGCAGTCC TGCCGTCATC ACTGACAAAG TA -            #ATCCCAGC   2100                                                                 - - TTGTCTGCCA TCCCCAAATT ATGTGGTCGC TGACCGGACC GAATGTTTCG TC -            #ACTGGCTG   2160                                                                 - - GGGAGAAACC CAAGGTACTT TTGGAGCTGG CCTTCTCAAG GAAGCCCAGC TC -            #CCTGTGAT   2220                                                                 - - TGAGAATAAA GTGTGCAATC GCTATGAGTT TCTGAATGGA AGAGTCCAAT CC -            #ACCGAACT   2280                                                                 - - CTGTGCTGGG CATTTGGCCG GAGGCACTGA CAGTTGCCAG GGTGACAGTG GA -            #GGTCCTCT   2340                                                                 - - GGTTTGCTTC GAGAAGGACA AATACATTTT ACAAGGAGTC ACTTCTTGGG GT -            #CTTGGCTG   2400                                                                 - - TGCACGCCCC AATAAGCCTG GTGTCTATGT TCGTGTTTCA AGGTTTGTTA CT -            #TGGATTGA   2460                                                                 - - GGGAGTGATG AGAAATAATT AATTGGACGG GAGACAG      - #                      - #    2497                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  pepti - #de                                        - -      (v) FRAGMENT TYPE:  internal fragment                                - -     (ix) FEATURE:                                                                  (A) OTHER INFORMATION: - #Tyr number 7 is 3-I-Tyr.                   - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:13:                   - - Pro Arg Lys Leu Tyr Asp Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  pepti - #de                                        - -      (v) FRAGMENT TYPE:  internal fragment                                - -     (ix) FEATURE:                                                                  (A) OTHER INFORMATION: - #Tyr number 5 is 3-I-Tyr.                   - -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:14:                   - - Pro Arg Lys Leu Tyr Asp Tyr                                              1               5                                                            __________________________________________________________________________

What is claimed is:
 1. A method of treating a disease in a patient inneed of antiangiogenesis therapy comprising adminstering to a human oranimal a therapeutically effective amount of a mammalian kringle 5peptide fragment.
 2. The method of claim 1 wherein said mammaliankringle 5 peptide fragment is selected from the group consisting ofhuman, murine, bovine, Rhesus monkey and porcine kringle 5 peptidefragment.
 3. The method of claim 2 wherein said kringle 5 peptidefragment is a human kringle 5 peptide fragment.
 4. The method of claim 1wherein said disease is selected from the group consisting of cancer,arthritis, macular degeneration and diabetic retinopathy.
 5. The methodof claim 4 wherein said disease is cancer.
 6. The method of claim 5wherein said disease is selected from primary and metastatic solidtumors, carcinomas, sarcomas, lymphomas, psoriasis and hemagiomas.
 7. Amethod of inhibiting endothelial cell proliferation, comprisingadministering to an endothelial cell an effective amount of a proteinhaving an amino acid sequence encoding a kringle 5 peptide fragment of aplasminogen molecule.
 8. The method of claim 7, wherein said plasminogenmolecule is human plasminogen.
 9. The method of claim 7, wherein saidprotein is selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
 7. 10. Amethod of treating a disease in a patient in need of antiangiogenesistherapy comprising administering to a human or animal a therapeuticallyeffective amount of a compound of the formula

    A-B-C-X-Y                                                  (I),

or a pharmaceutically acceptable salt, ester or prodrug thereof, whereinA is absent or a nitrogen protecting group; Y is absent or a carboxylicacid protecting group; B is absent or is from 1 to about 197naturally-occurring amino acid residues having substantial sequencehomology to the corresponding amino acid sequence from about amino acidposition 334 to amino acid position 530 of SEQ ID NO: 1; C is R¹ -R² -R³-R⁴ whereinR¹ is lysyl, R² is leucyl or arginyl, R³ is tyrosyl,3-I-tyrosyl or phenylalanyl; and R⁴ is aspartyl, and X is absent or isfrom 1 to about 12 naturally occurring amino acid residues havingsubstantial sequence homology to the corresponding amino acid sequencefrom amino acid position 535 to about amino acid position 546 of SEQ IDNO:
 1. 11. The method of claim 10 wherein B is present and A, C, X, andY are as defined therein.
 12. The method of claim 10 wherein A and Y areabsent and B, C, and X are as defined therein.